Packaging systems for human recombinant adenovirus to be used in gene therapy

ABSTRACT

Improved methods and products based on adenoviral materials which can advantageously be used in, for instance, gene therapy. In one aspect, an adenoviral vector is provided which has no overlap with a suitable packaging cell line, the packaging cell line comprising another aspect of the invention. This combination excludes the possibility of homologous recombination, thereby excluding the possibility of the formation of replication competent adenovirus. In another aspect, an adenovirus based helper construct which by its size is incapable of being encapsulated is disclosed. This helper virus can be transferred into any suitable host cell making it a packaging cell. Additionally, a number of useful mutations to adenoviral based materials and combinations of such mutations are disclosed. Furthermore, a method of intracellular amplification is provided.

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application is a divisional application of co-pending U.S. Ser. No. 09/065,752, filed Apr. 24, 1998, the contents of which are incorporated herein by this reference.

BACKGROUND OF THE INVENTION

[0002] 1. Technical Field

[0003] The invention relates to the field of recombinant DNA technology, especially the field of gene therapy. More specifically, the invention relates to gene therapy using materials derived from adenovirus, in particular, human recombinant adenovirus. It particularly relates to novel virus derived vectors and novel packaging cell lines for vectors based on adenoviruses.

[0004] 2. Background

[0005] Gene therapy is a relatively recently developed concept for which a wide range of applications can be and have been envisaged. In gene therapy, a molecule carrying genetic information is introduced into some or all cells of a host, as a result, the genetic information is added to the host in a functional format. Gene therapy also includes the treatment of genetic disorders by providing the genetic information for supplementing a protein or other substance that is, through the genetic disorder, not present or present in insufficient amounts in the host, the treatment of tumors and (other) acquired diseases such as (auto) immune diseases, infections, or other processes. The genetic information added may be a gene or a derivative of a gene, such as a cDNA, that encodes a protein. In this case, the functional format means that the protein can be expressed by the machinery of the host cell. The genetic information can also be a sequence of nucleotides complementary to a sequence of nucleotides (be it DNA or RNA) present in the host cell. The functional format in this case is that the added DNA (nucleic acid) molecule, or copies made thereof in situ, is capable of base pairing with the complementary sequence present in the host cell.

[0006] Thus, there are basically three different approaches in gene therapy, the first directed toward compensating a deficiency present in a (mammalian) host; the second directed toward the removal or elimination ofunwanted substances (organisms or cells) and the third directed towards application of a recombinant vaccine (tumors or foreign microorganisms).

[0007] Adenoviruses carrying deletions have been proposed as suitable vehicles for the purpose of gene therapy. Adenoviruses are non-enveloped DNA viruses. Gene transfer vectors derived from adenoviruses (so called adenoviral vectors) have a number of features that make them particularly useful for gene transfer. For example, the biology of the adenovirus is characterized in detail, is not associated with severe human pathology, is extremely efficient in introducing its DNA into a host cell, can infect a wide variety of cells and has a broad host range, can be produced in large quantities with relative ease, and can be rendered replication defective by deletions in the early region 1 (E1) of the viral genome.

[0008] The genome of adenovirus (“Ad”) is a linear double-stranded DNA molecule of approximately 36,000 base pairs (“bp”) with the 55-kDa terminal protein covalently bound to the 5′ terminus of each strand. The Ad DNA contains identical Inverted Terminal Repeats (“ITRs”) of about 100 bp with the exact length depending on the serotype. The viral origins of replication are located within the ITRs exactly at the genome ends. DNA synthesis occurs in two stages. First, the replication proceeds by strand displacement, generating a daughter duplex molecule and a parental displaced strand. The displaced strand is single stranded and can form a so called “panhandle” intermediate, which allows replication initiation and generation of a daughter duplex molecule. Alternatively, replication may proceed from both ends of the genome simultaneously, obviating the requirement to form the panhandle structure. The replication is summarized in FIG. 14 adapted from Lechner et al., (1977) J. Mol. Biol. 174:493-510.

[0009] During the productive infection cycle, the viral genes are expressed in two phases: the early phase, which is the period up to viral DNA replication, and the late phase, which coincides with the initiation of viral DNA replication. During the early phase, only the early gene products, encoded by regions E1, E2, E3 and E4, are expressed. The early gene products carry out a number of functions that prepare the cell for synthesis of viral structural proteins (Berk, A. J. (1986) Ann. Rev. Genet. 20: 45-79). During the late phase, the late viral gene products are expressed in addition to the early gene products and host cell DNA and protein synthesis are shut off. Consequently, the cell becomes dedicated to the production of viral DNA and of viral structural proteins (Tooze, J. DNA Tumor Viruses (revised), Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y., (1981)).

[0010] The E1 region of adenovirus is the first region of adenovirus expressed after infection of the target cell. This region consists of two transcriptional units, the E1A and E1B genes, both of which are required for oncogenic transformation of primary (embryonal) rodent cultures. The main functions of the E1A gene products are to induce quiescent cells to enter the cell cycle and resume cellular DNA synthesis, and to transcriptionally activate the E1B gene and the other early regions (E2, E3 and E4) of the viral genome. Transfection of primary cells with the EIA gene alone can induce unlimited proliferation (immortalization), but does not result in complete transformation. However, expression of E1A in most cases results in induction of programmed cell death (apoptosis), and only occasionally immortalization is obtained (Jochemsen et al., EMBO J 6:3399-3405 (1987)). Co-expression of the EIB gene is required to prevent induction of apoptosis and for complete morphological transformation to occur. In established immortal cell lines, high level expression of E1A can cause complete transformation in the absence of E1B (Roberts et al., J. Virol. 56:404-413 (1985)).

[0011] The E1 B encoded proteins assist E1A in redirecting the cellular functions to allow viral replication. The E1B 55 kD and E4 33kD proteins, that form a complex that is essentially localized in the nucleus, function in inhibiting the synthesis of host proteins and in facilitating the expression of viral genes. Their main influence is to establish selective transport of viral mRNAs from the nucleus to the cytoplasm, concomitantly with the onset of the late phase of infection. The E1B 21 kD protein is important for correct temporal control of the productive infection cycle, thereby preventing premature death of the host cell before the virus life cycle has been completed. Mutant viruses incapable of expressing the E1B 21 kD gene product exhibit a shortened infection cycle that is accompanied by excessive degradation of host cell chromosomal DNA (deg-phenotype) and an enhanced cytopathic effect (cyt-phenotype) (Telling et al., J. Virol 68:541-7 (1994)). The deg and cyt phenotypes are suppressed when the E1A gene is also mutated, indicating that these phenotypes are a function of E1A (White et al., J. Virol. 62:3445-3454 (1988)). Furthermore, the E1B 21 kDa protein slows down the rate by which E1A switches on the other viral genes. It is not yet known through which mechanisms E1B 21 kD quenches these E1A dependent functions.

[0012] Vectors derived from human adenoviruses, in which at least the E1 region has been deleted and replaced by a gene of interest, have been used extensively for gene therapy experiments in the pre-clinical and clinical phase, and all adenoviral vectors currently used in gene therapy have a deletion in the E1 region where novel genetic information can be introduced. The E1 deletion renders the recombinant virus replication defective (Stratford-Perricaudet et al., (1991) pp.51-61.; and O. Cohen-Adenaur and M. Boiron (Eds): Human Gene Transfer, John Libbey Eurotext).

[0013] In contrast to retroviruses, for example, adenoviruses do not integrate into the host cell's genome, are able to infect non-dividing cells, and are able to efficiently transfer recombinant genes in vivo (Brody et al., (1994) Ann N Y Acad Sci. 716:90-101). These features make adenoviruses attractive candidates for in vivo gene transfer of, for example, suicide or cytokine genes into tumor cells. However, a problem associated with current recombinant adenovirus technology is the possibility of unwanted generation of replication competent adenovirus (“RCA”) during the production of recombinant adenovirus (Lochmüller et al., (1994) Hum. Gene Ther. 5:1485-1492; Imler et al., (1996) Gene Ther. 3:75-84). This is caused by homologous recombination between overlapping sequences from the recombinant vector and the adenovirus constructs present in the complementing cell line, such as the 293 cells (Graham et al., (1977) J. Gen. Virol. 36:59-72). RCA in batches to be used in clinical trials is unwanted because RCA i) replicates in an uncontrolled fashion; ii) can complement replication defective recombinant adenovirus, causing uncontrolled multiplication of the recombinant adenovirus; and iii) induces significant tissue damage and hence strong pathological side effects (Lochmüller et al., (1994) Hum. Gene Ther. 5:1485-1492). Therefore, batches to be used in clinical trials should be proven free of RCA (Ostrove, J. M. (1994) Cancer Gene Ther. 1: 125-131).

[0014] An additional problem associated with the use of recombinant adenoviral vectors is the host defense reaction against treatment with adenovirus. Briefly, recombinant adenoviruses are deleted for the E1 region (see, above). The adenoviral E1 products trigger the transcription of the other early genes (E2, E3, E4), which consequently activate expression of the late virus genes. Therefore, it was generally thought that E1-deleted vectors would not express any other adenoviral genes. However, recently it has been demonstrated that some cell types are able to express adenoviral genes in the absence of E1 sequences. This indicates that some cell types possess the machinery to drive transcription of adenoviral genes. In particular, it was demonstrated that such cells synthesize E2A and late adenoviral proteins. In a gene therapy setting, this means that transfer of the therapeutic recombinant gene to somatic cells not only results in expression of the therapeutic protein but may also result in the synthesis of viral proteins. Cells that express adenoviral proteins are recognized and killed by cytotoxic T Lymphocytes, which both eradicates the transduced cells and causes inflammation (Bout et al., (1994a) Gene Therapy 1:385-394; Engelhardt et al., (1993) Human Gene Therapy 4:759-769; Simon et al., (1993) Human Gene Therapy 4:771-780).

[0015] As this adverse reaction hampers gene therapy, several solutions to this problem have been suggested, such as; using immunosuppressive agents after treatment, retaining the adenoviral E3 region in the recombinant vector (see, EPO Patent Application EP 9520221 B) or using temperature sensitive (“ts”) mutants of human adenovirus, which have a point mutation in the E2A region (PCT International Patent Application WO/28938). However, these strategies to circumvent the immune response have their limitations. The use of ts mutant recombinant adenovirus diminishes the immune response to some extent, but is less effective in preventing pathological responses in the lungs (Engelhardt et al., (1994a) Human Gene Ther. 5:1217-1229). The E2A protein may induce an immune response by itself and it plays a pivotal role in the switch to the synthesis of late adenoviral proteins. Therefore, it is attractive to make recombinant adenoviruses that are mutated in the E2 region, rendering the viruses temperature sensitive (“ts”), as has been identified in PCT International Patent Application WO/28938.

[0016] A major drawback of this system is the fact that, although the E2 protein is unstable at the non-permissive temperature, the immunogenic protein is still synthesized. In addition, the unstable protein activates late gene expression, albeit to a low extent. ts125 mutant recombinant adenoviruses have been tested, and prolonged recombinant gene expression was reported (Yang et al., (1994b) Nat Genet. 7:362-369; Engelhardt et al., (1994a) Hum. Gene Ther. 5:1217-1229; Engelhardt et al., (1994b) Proc Natl Acad Sci USA 91:6196-200; Yang et al., (1995) J. Virol. 69:2004-2015). However, pathology in the lungs of cotton rats was still high (Engelhardt et al., (1994a) Human Gene Ther. 5:1217-1229), indicating that the use of ts mutants results in only a partial improvement in recombinant adenovirus technology. Others (Fang et al., (1996) Gene Ther. 3:217-222), did not observe prolonged gene expression in mice and dogs using ts125 recombinant adenovirus. An additional difficulty associated with the use of ts125 mutant adenoviruses is that a high frequency of reversion is observed. These revertants are either real revertants or the result of second site mutations (Kruij er et al., (1983) Virology 124:425-433; Nicolas et al., (1981) Virology 108:521-524). Both types of revertants have an E2A protein that functions at normal temperature and therefore have similar toxicity as the wild-type virus.

[0017] E1-deleted recombinant adenoviruses are usually made by one of the following methods. In the first method, adenovirus DNA, be it wild type (wt) or E1-and/or E3-deleted, is digested with a restriction enzyme, for example, ClaI, to remove the left ITR, packaging signal and at least part of the E1 sequences, and the remaining adenovirus genome fragment (1) is purified. Cotransfection of (1) with a linearized adapter construct (2) containing the left ITR, packaging signal, an expression cassette with the gene of interest, and adenovirus sequences overlapping with (1) in a cell line complementing for E1 functions (packaging cell line), will give rise to recombinant adenovirus particles by intra-cellular homologous recombination. Alternatively, an adapter construct (3) containing the left ITR, packaging signal, and an expression cassette with the gene of interest, is such that it can be ligated to the adenovirus DNA fragment (1) followed by transfection into packaging cells.

[0018] The disadvantage of these methods is that the purification of (1) is laborious and incomplete digestion of wt DNA results in introduction of wt adenovirus into the culture leading to contamination. An approach to circumvent this problem has included the construction of clone pHBG10 described by Bett et al., (1994) Natl. Acad. Sci. USA 91:8802-8806. This plasmid clone contains adenovirus 5 (“Ad5”) sequences with a deletion of the packaging signal and part of the E1 region and with the viral ITRs attached to each other. However, this clone includes adenovirus sequences that are also present in E1-complementing cell lines, including those of the present invention (see, EPO Patent Application EP95201611.1). Furthermore, since the ITRs are attached to each other, the clone cannot be linearized resulting in less efficient recombination with the E1 substitution plasmid.

[0019] In the second method, recombinant adenoviruses are constructed either by homologous recombination in bacteria (Chartier et al., (1996) J. Virol. 70, No.7:4805-4810; Croozet et al., (1997) Proc. Natl. Acad. Sci. USA 94:1414-1419) or by cloning into cosmid vectors (Fu et al., (1997) Hum. Gene Ther. 8:1321-1330) and subsequently transfecting into an E1 complementing cell line. The disadvantage of this method is that it demands extensive analysis of each generated clone (˜35 kb) by restriction enzyme digestion before transfection to exclude deletions that occurred due to recombination in the bacteria. In addition, the use of cloned adenovirus sequences does not solve the problem of sequence overlap between commonly used packaging cells and recombinant viruses leading to production of RCA during propagation.

[0020] The third method uses a two-step gene replacement technique in yeast, starting with a complete adenovirus genome (Ad2); (Ketner et al., (1994) Proc. Natl. Acad. Sci. USA 91:6186-6190) cloned in a Yeast Artificial Chromosome (“YAC”) and aplasmid containing adeno sequences to target a specific region in the YAC clone, an expression cassette for the gene of interest and a positive and negative selectable marker. This method requires yeast technology and extensive analysis of each new recombinant clone (even more cumbersome than the above described method, due to the large size of YACs).

[0021] The fourth method uses a cosmid clone (pAdexlw; Miyake et al., (1996) Medical Sciences 93:1320-1324) that carries the Ad5 sequence with deletions in the E1 and E3 sequences. This clone has a unique restriction site replacing part of the E1 region that allows insertion of a foreign expression cassette. For generating recombinant adenoviruses, a DNA-terminal protein complex (DNA-TPC) is isolated from cells infected with a replication competent adenovirus (“RCA”) Ad-dlX (wt Ad5 with an XbaI deletion in the E3 region). This DNA is digested with EcoT22I, to remove the 5′ part of the DNA, and co-transfected with the cosmid cloned into E1 complementing cells. Intracellular recombination generates the recombinant virus (Miyake et al., (1996) Medical Sciences 93:1320-1324). This method has the disadvantage that replication competent viral DNA is used and that the E1 deletion in the cosmid clone is not enough to remove all overlap with E1 sequences in currently used packaging cell lines including those used in the present invention.

[0022] Thus, current methods to generate RCA-free recombinant adenoviruses have several disadvantages, including the risk of introducing wild-type viruses in the culture, instability of cloned adenovirus sequences, the necessity to check the complete ˜35 kb recombinant clone by restriction analysis for each new virus to be generated, and the system being suitable only for E1-deleted recombinant adenoviruses and much more laborious for use with recombinant adenoviruses including E3 substitutions. Furthermore, despite the use of cloned adenovirus DNA in some of the methods, extensive overlap with adenovirus sequences present in commonly used packaging cells, like 293 and 911 cells, does not solve the problem of appearance of RCA due to homologous recombination during propagation of the virus. Therefore, a need persists for methods and means to produce RCA-free recombinant adenovirus preparations that solve the disadvantages of prior art methods and means discussed above. Gene addition is currently by far the most widely applied gene therapy technique. This is primarily due to the fact that homologous recombination is very inefficient and for homologous recombination, relatively large DNA fragments are required for which no suitable vector systems were available. Thus, there is currently an unmet need for vector systems that efficiently introduce large nucleic acid molecules into mammalian cells.

[0023] Recombinant adenoviruses are able to efficiently transfer recombinant genes to the rat liver and airway epithelium of rhesus monkeys (Bout et al., (1994b) Human Gene Therapy 5:3-10; Bout et al., (1994a) Gene Therapy 1:385-394). In addition, (Vincent et al., (1996) J. Neurosurg 85:648-654; Vincent et al., (1996b) Hum. Gene Ther. 7:197-205) and others (see, e.g., Haddada et al., (1993) Hum. Gene Ther. 4:703-11) have observed an efficient in vivo adenovirus -mediated gene transfer to a variety of tumor cells in vitro and to solid tumors in animal models (lung tumors, glioma) and human xenografts in immunodeficient mice (lung) in vivo (reviewed by Blaese et al., Cancer Gene Ther. 2:291-297).

[0024] Generation ofminimal adenoviral vectors has been disclosed in PCT International Patent Application WO 94/12649. The method described exploits the function of the protein IX for the packaging of minimal adenoviral vectors (Pseudo Adenoviral Vectors (“PAV”) in the terminology of WO 94/12649). PAVs are produced by cloning an expression plasmid with the gene of interest between the left-hand (including the sequences required for encapsulation) and the right-hand adenoviral ITRs. The PAV is propagated in the presence of a helper virus. Encapsulation of the PAV is preferred compared to the helper virus because the helper virus is partially defective for packaging (either by virtue of mutations in the packaging signal or by virtue of its size (virus genomes greater than 37.5 kb package inefficiently)). In addition, it proposes that, in the absence of the protein IX gene, the PAV will be preferentially packaged. However, neither of these mechanisms appear to be sufficiently restrictive to allow packaging of only PAVs/minimal vectors. The mutations proposed in the packaging signal diminish packaging, but do not provide an absolute block as the same packaging activity is required to propagate the helper virus. Also neither an increase in the size of the helper virus nor the mutation of the protein IX gene will ensure that PAV is packaged exclusively. Thus, the method described in WO 94/12649 is unlikely to be useful for the production of helper-free stocks of minimal adenoviral vectors PAVs.

DESCRIPTION OF THE INVENTION

[0025] The invention includes means and methods for generating adenoviral vectors. In one aspect, the invention provides novel compositions and methods producing recombinant adenoviruses, not only E1-deleted, but also minimal adenoviruses that are free of replication competent adenoviruses. One aspect of the invention provides a recombinant nucleic acid comprising a nucleotide sequence based on or derived from an adenovirus, the nucleotide sequence comprising a functional encapsulating signal and two functional Inverted Terminal Repeats or functional fragments or derivatives thereof, the recombinant nucleic acid comprising no functional adenoviral genes and no overlapping sequences which would allow for homologous recombination leading to replication competent virus in a cell into which the recombinant nucleic acid is transferred.

BRIEF DESCRIPTION OF THE DRAWINGS

[0026] The hereinafter described drawings may help one in understanding the invention:

[0027]FIG. 1 depicts construction of pBS.PGK.PCRI.

[0028] pBS.PGK.PCRI encodes the human phosphoglycerate kinase promoter (“PGK”) operatively linked to Ad5 E1 nucleotides 459-916. To construct this plasmid, Ad5 nucleotides 459-916 were PCR amplified with primers Ea-1 and Ea-2, (Table 1) digested with Cla I and cloned into the Cla I-EcoRV sites of pBluescript (Stratagene), resulting in pBS.PCRI. The PGK promoter was excised from pTN by complete digestion with Sca I and partial digestion with EcoR I and cloned into the corresponding sites of pBS.PCRI, resulting in pBS.PGK.PCRI.

[0029]FIG. 2 depicts construction of pIG.E1A.E1B.X.

[0030] pIG.E1A.E1B.X encodes Ad5 nucleotides 459-5788 (E1A and E1B regions) operatively linked to the human PGK promoter. pIG.E1A.E1B.X also encodes Ad5 pIX protein. pIG.E1A.E1B.X was constructed by replacing the Sca I-BspE I fragment of pAT-X/S with the corresponding fragment of pBS.PGK.PCRI.

[0031]FIGS. 3A and 3B depict construction of pIG.E1A.NEO.

[0032] pIG.E1A.NEO encodes Ad5 nucleotides 459-1713 operatively linked to the human PGK promoter. Also encoded is the E1B promoter functionally linked to the neomycin resistance gene (Neo^(R)) and the hepatitis B virus (“HBV”) poly(A) signal. In this construct, the AUG codon of the E1B 21 kDa protein functions as the initiation codon of Neo^(R). To construct this plasmid, the E1B promoter and initiation codon (ATG) of the E1B 21 kDa protein were PCR amplified with primers Ea-3 and Ep-2, where Ep-2 introduces an Nco I site (5′-CCATGG) at the 21 kDa protein initiation codon. The PCR product (PCRII) was digested with HpaI and NcoI and ligated into the corresponding sites of pAT-X/S, producing pAT-X/S-PCR2. The Nco I-Stu I fragment of pTN, containing the Neo^(R) and a portion of the HBV poly(A) site were ligated into the Nco I-Nru I sites of pAT-X/S-PCR2, producing pAT-PCR2-NEO. The HBV poly(A) signal was completed by replacing the Sca I-Sal I fragment of pAT-PCR2-NEO with the corresponding fragment of pTN, producing pAT.PCR2.NEO.p(A), and replacing the Sca I-Xba I fragment of pAT.PCR2.NEO.p(A) with the corresponding fragment of pIG.E1A.E1B.X, producing pIG.E1A.NEO.

[0033]FIG. 4 depicts construction of pIG.E1A.E1B.

[0034] pIG.E1A.E1B contains the Ad5 nucleotides 459-3510 (E1A and E1B proteins) operatively linked to the PGK promoter and HBV poly(A) signal. This plasmid was constructed by PCR amplification of the N-terminal amino acids of the E1B 55 kD protein with primers Eb-1 and Eb-2, which introduces an Xho I site, digested with Bgl II and cloned into the Bgl II-Nru I sites of pAT-X S, producing pAT-PCR3. The Xba I-Xho I fragment of pAT-PCR3 was replaced with the Xba I-SalI fragment (containing the HBV poly(A) site) of pIG.E 1A.NEO to produce pIG.E1A.E1B.

[0035]FIG. 5 depicts construction of pIG.NEO.

[0036] pIG.NEO contains the Neo^(R) operatively linked to the E1B promoter. pIG.NEO was constructed by ligating the Hpa I-Sca I fragment of pIG.E1A.NEO which contains the E1B promoter and Neo^(R) into the EcoR V-Sca I sites of pBS.

[0037]FIG. 6 is a graphical overview of available adenovirus packaging cells assessing their capacity to transform primary kidney cells.

[0038] It generally depicts the transformation of primary baby rat kidney (“BRK”) cells by adenovirus packaging constructs. Subconfluent dishes of BRK cells were transfected with 1 or 5 μg of with either pIG.NEO, pIG.E1A.NEO, pIG.E1A.E1B, pIG.E1A.E1B.X, pAd5XhoIC, or pIG.E1A.NEO plus pDC26, which expresses the Ad5 E1B gene under control of the SV40 early promoter. Three weeks post-transfection, foci were visible, cells were fixed, Giemsa stained and the foci counted. The results shown are the average number of foci per 5 replicate dishes.

[0039]FIG. 7 is western blot analysis of A549 clones transfected with pIG.E1 A.NEO and human embryonic retinoblasts (“HER” cells) transfected with pIG.E1A.E1B (PER clones).

[0040] Expression of Ad5 E1A and E1B 55 kD and 21 kD proteins in transfected A549 cells and PER cells was determined by Western blot with mouse monoclonal antibodies (Mab) M73 which recognizes E1A gene products and Mabs AIC6 and C1G11, which recognize the E1B 55 kDa and 21 kDa proteins, respectively. Mab binding was visualized using horseradish peroxidase-labeled goat anti-mouse antibody and enhanced chemiluminesence. 293 and 911 cells served as controls.

[0041]FIG. 8 is a southern blot analysis of 293, 911 and PER cell lines.

[0042] Cellular DNA was extracted, Hind III digested, electrophoresed and transferred to Hybond N+ membranes (Amersham). Membranes were hybridized to radio-labeled probes generated by random priming of the Ssp I-Hind in fragment of pAd5.SalB (Ad5 nucleotides 342-2805).

[0043]FIG. 9 illustrates the transfection efficiency ofPER.C3, PER.C5, PER.C6 and 911 cells.

[0044] Cells were cultured in 6-well plates and transfected in duplicate with 5 μg pRSV.lacZ by calcium-phosphate co-precipitation. Forty-eight hours post-transfection, cells were stained with X-Gal and blue cells were counted. Results shown are the mean percentage of blue cells per well.

[0045]FIG. 10 depicts construction of adenoviral vector, pMLPI.TK.

[0046] pMLPI.TK was designed to have no sequence overlap with the packaging construct pIG.E1A.E1B. pMLPI.TK was derived from pMLP.TK by deletion of the region of sequence overlap with pIG.E1A.E1B and deletion of non-coding sequences derived from lacZ. SV40 poly(A) sequences of pMLP.TK were PCR amplified with primers SV40-1, which introduces a BamH I site and SV40-2, which introduces a Bgl II site. pMLP.TK Ad5 sequences 2496 to 2779 were PCR amplified with primers Ad5-1, which introduces a Bgl II site and Ad5-2. Both PCR products were Bgl II digested, ligated, and PCR amplified with primers SV40-1 and Ad5-2. This third PCR product was BamH I and Afl III digested and ligated into the corresponding sites of pMLP.TK, producing pMLPI.TK.

[0047]FIGS. 11 A and B. show that the new adenovirus packaging constructs do not have sequence overlap with new adenoviral vectors.

[0048]FIG. 11A is a packaging system based on primary cells.

[0049]FIG. 11B is a packaging system based on established cell lines: transfection with E1 a and selection with G418. Regions of sequence overlap between the packaging construct, pAd5XhoIC expressed in 911 cells and adenoviral vector, pMLP.TK, that can result in homologous recombination and the formation of replication competent adenovirus are shown (FIG. 11A). In contrast, there are no regions of sequence overlap between the new packaging construct, pIG.E1A.E1B, expressed in PER.C6 cells, and the new adenoviral vector, pMLPI.TK, (FIG. 11A) or between the new packaging construct, pIG.E1A.NEO and the new adenoviral vector pMLPI.TK (FIG. 11B) that can result in homologous recombination and the formation of RCA.

[0050]FIG. 12 depicts the generation of recombinant adenovirus IG.Ad.MLPI.TK.

[0051] Recombinant adenovirus, IG.Ad.MLPI.TK, was generated by co-transfection of 293 cells, with Sal I linearized pMLPI.TK and the right arm of Cla I digested, wild-type Ad5 DNA. Homologous recombination between linearized pMLPI.TK and wild-type Ad5 DNA produces IG.Ad.MLPI.TK DNA, which contains an E1 deletion of nucleotides 459-3510. 293 cells transcomplement the deleted Ad5 genome, thereby, permitting replication of the IG.Ad.MLPI.TK DNA and its packaging into virus particles.

[0052]FIG. 13 depicts the rationale for the design of adenovirus-derived recombinant DNA molecules that duplicate and replicate in cells expressing adenovirus replication proteins.

[0053] A diagram of the adenovirus double-stranded DNA genome indicating the approximate locations of E1, E2, E3, E4, and L regions is shown. The terminal polypeptide (“TP”) attached to the 5′-termini is indicated by closed circles. The right arm of the adenovirus genome can be purified by removal of the left arm by restriction enzyme digestion. Following transfection of the right arm into 293 or 911 cells, adenoviral DNA polymerase (white arrow) encoded on the right arm, will produce only single-stranded forms. Neither the double-stranded or single-stranded DNA can replicate because they lack an ITR at one terminus. Providing the single-stranded DNA with a sequence that can form a hairpin structure at the 3′-terminus that can serve as a substrate for DNA polymerase will extend the hairpin structure along the entire length of the molecule. This molecule can also serve as a substrate for a DNA polymerase but the product is a duplicated molecule with ITRs at both termini that can replicate in the presence of adenoviral proteins.

[0054]FIG. 14 depicts adenovirus genome replication.

[0055] The adenovirus genome is shown in the top left. The origins or replication are located within the left and right ITRs at the genome ends. DNA replication occurs in two stages. Replication proceeds from one ITR generating a daughter duplex and a displaced parental single-strand which is coated with adenovirus DNA binding protein (DBP, open circles) and can form a panhandle structure by annealing of the ITR sequences at both termini. The panhandle is a substrate for DNA polymerase (Pol: white arrows) to produce double-stranded genomic DNA. Alternatively, replication proceeds from both ITRs, generating two daughter molecules, thereby, obviating the requirement for a panhandle structure.

[0056]FIG. 15 depicts a potential hairpin conformation of a single-stranded DNA molecule that contains the HP/asp sequence.

[0057] Asp718 I digestion ofpICLha, containing the cloned oligonucleotides, HP/asp1 and HP/asp2 yields a linear double-stranded DNA with an Ad5 ITR at one terminus and the HP/asp sequence at the other terminus. In cells, expressing the adenoviral E2 region, a single-stranded DNA is produced with an Ad5 ITR at the 5′-terminus and the hairpin conformation at the 3′-terminus. Once formed, the hairpin can serve as a primer for cellular and/or adenovirus DNA polymerase to convert the single stranded DNA to double stranded DNA.

[0058]FIG. 16 is a diagram of pICLhac.

[0059] pICLhac contains all the elements of pICL (FIG. 19) but also contains in the Asp 718 site, the HP/asp sequence in an orientation that will produce the hairpin structure shown in FIG. 15, following linearization by Asp 718 digestion and transfection into cells expressing adenoviral E2 proteins.

[0060]FIG. 17 is a diagram of pICLhaw.

[0061] pICLhaw is identical to pICLhac (FIG. 16) with the exception that the inserted HP/asp sequence is in the opposite orientation.

[0062]FIG. 18 is a schematic representation of pICLI.

[0063] pICLI contains all the elements of pICL (FIG. 19) but also contains in the Asp 718 site, an Ad5 ITR.

[0064]FIG. 19 is a diagram of pICL.

[0065] pICL is derived from the following: (i) nucleotides 1-457, Ad5 nucleotides 1-457 including the left ITR, (ii) nucleotides 458-969, human CMV enhancer and immediate early promoter, (iii) nucleotides 970-1204, SV40 19S exon and truncated 16/19S intron, (iv) nucleotides 1218-2987, firefly luciferase gene, (v) nucleotides 3018-3131, SV40 tandem polyadenylation signals from the late transcript, (vi) nucleotides 3132-5620, pUC12 sequences including an Asp718 site, and (vii) ampicillin resistance gene in reverse orientation.

[0066]FIG. 20 shows a schematic overview of the adenovirus fragments cloned in pBr322 (plasmid) or pWE15 (cosmid) derived vectors.

[0067] The top line depicts the complete adenovirus genome flanked by its ITRs (filled rectangles) and with some restriction sites indicated. Numbers following restriction sites indicate approximate digestion sites (in kb) in the Ad5 genome.

[0068]FIG. 21 is a drawing of adapter plasmid pAd/L420-HSA

[0069]FIG. 22 is a drawing of adapter plasmid pAd/Clip

[0070]FIG. 23 schematically presents the generation of recombinant adenoviruses using a plasmid-based system.

[0071] In the top, the genome organization of Ad5 is given with filled boxes representing the different early and late transcription regions, and flanking ITRs. The middle presents the two DNAs used for a single homologous recombination and, after transfection into packaging cells, leading to the recombinant virus (represented at the bottom).

[0072]FIG. 24 is a drawing of minimal adenoviral vector pMV/L420H.

[0073]FIG. 25 schematically presents the cloning steps for the generation of the helper construct pWE/AdA5′.

[0074]FIG. 26 provides evidence for SV40-LargeT/ori mediated replication of large adenoviral constructs in Cos-1 cells.

[0075] Part A: Schematic presentation of construct pWE/Ad.Δ5′ and the location of the SV40 ori sequence and the fragments used to prepare probes. Part B: Autoradiogram of the Southern blot hybridized to the adenovirus probe. Part C: Autoradiogram of the Southern blot hybridized to the pWE probe.

[0076] Lanes 1, marker lane: λ DNA digested with EcoR I and Hind III. Lane 4 is empty. Lanes 2, 5, 7, 9, 11, 13, 15 and 17 contain undigested DNA and Lanes 3, 6, 8, 10, 12, 14, 16 and 18 contain Mbo I digested DNA. All lanes contain DNA from Cos-l cells as described in the text transfected with pWE.pac (lanes 2 and 3), pWE/Ad.Δ5′ construct #1 (lanes 5 and 6), #5 (lanes 7 and 8) and #9 (lanes 9 and 10), pWE/Ad.AflII-rITR (lanes 11 and 12), pMV/CMV-LacZ (lanes 13 and 14), pWE.pac digested with Pac I (lanes 15 and 16) or pWE/Ad.AflII-rITR digested with Pac I (lanes 17 and 18). Arrows point at the expected positive signal of 1416 bp (B) and 887 bp (C).

DETAILED DESCRIPTION OF THE INVENTION

[0077] Novel compositions and methods are provided for producing recombinant adenoviruses, not only E1-deleted, but also minimal adenoviruses that are free of replication competent adenoviruses. The compositions include constructs suitable for the generation of double insert viruses. The system provided by the invention for generating, for example, E1-deleted adenoviruses consisting of two nucleic acid molecules. A first of which is a relatively small and easy to manipulate adapter plasmid containing at least, in an operable configuration, the left ITR, the packaging signal, an expression cassette with the nucleic acid molecule of interest and adenovirus sequences homologous to a part of a second molecule that includes one, or two or more partially overlapping, nucleic acid molecules that include at least all adenovirus sequences necessary for replication and packaging, and the packaging cells of the invention described infra. Co-transfection of the two nucleic acid molecules into the packaging cells leads to homologous recombination between the overlapping sequences in said two nucleic acid molecules. Homologous recombination generates a recombinant viral DNA that is capable of replicating and propagating on the packaging cells. The nucleic acid molecules have no sequence overlap with the E1 complementing sequences in packaging cells that can lead to the formation of RCA. Preferably, at least one of the ITRs on the nucleic acid molecules is flanked by a restriction enzyme recognition site not present in the adenoviral sequences so that the ITR can be made free from vector sequences by digestion of the DNA with that restriction enzyme. In this way, initiation of replication occurs more efficiently.

[0078] The system of the present invention also greatly facilitates the production of RCA-free recombinant adenoviruses with further modifications in the adenovirus genome, including, for example, modifications in the coding regions for the penton base protein or fiber protein or E2A protein.

[0079] In one embodiment of the invention, we have solved the problem of RCA production in that packaging cells have been developed having no overlapping sequences with a new basic vector, and thus are suitable for safe, large-scale production of recombinant adenoviruses.

[0080] In another embodiment of the present invention, we therefore delete E2A coding sequences from the recombinant adenovirus genome and transfect these E2A sequences into the (packaging) cell lines containing E1 sequences to complement recombinant adenoviral vectors. Major hurdles to this approach are that E2A should be expressed to very high levels and that E2A protein is very toxic to cells.

[0081] The current invention, in yet another aspect, discloses use of the ts 125 mutant E2A gene, which produces a protein that is unable to bind DNA sequences at the non permissive temperature. High levels of this protein may be maintained in the cells (because it is non-toxic at the particular temperature) until the switch to the permissive temperature is made. This can be combined with placing the mutant E2A gene under the direction of an inducible promoter, including, but not limited to tet, methallothionein, steroid inducible promoter, retinoic acid β-receptor or other inducible systems. In yet another embodiment of the invention, the use of an inducible promoter to control the moment of production of toxic wild-type E2A is disclosed.

[0082] Two salient additional advantages of E2A-deleted recombinant adenovirus are the increased capacity to harbor heterologous sequences and the permanent selection for cells that express the mutant E2A. The second advantage relates to the high frequency of reversion of ts125 mutation: when reversion occurs in a cell line harboring ts125 E2A, this will be lethal to the cell. Therefore, a permanent selection exists for those cells that express the ts125 mutant E2A protein. In addition, as we in one embodiment of the invention generate E2A-deleted recombinant adenovirus, we should not have the problem of reversion in the generated adenoviruses.

[0083] In yet another aspect of the invention, the use of non-human cell lines as packaging cell lines is disclosed. For GMP production of clinical batches of recombinant viruses, it is desirable to use a cell line that has been used widely for production of other biotechnology products. Most of the latter cell lines are from monkey origin, that have been used to produce, for example, vaccines. These cells cannot be used directly for producing recombinant human adenovirus, as human adenovirus cannot replicate in cells of monkey origin or only replicates at low levels. A block in the switch of early to late phase of adenovirus lytic cycle underlies the defective replication. However, host range (“hr”) mutations in the human adenovirus genome are described (hr400-404) that allow replication of human viruses in monkey cells. These mutations reside in the gene encoding E2A protein (Klessig et al., (1979) Cell 17:957-966; Klessig et al., (1984) Virus Res. 1:169-188; Rice et al., (1985) J. Virol. 56:767-778) (Klessig et al., (1984) Virus Res. 1:169-188). Moreover, mutant viruses have been described that harbor both the hr and temperature-sensitive ts125 phenotype (Brough et al., (1985) J. Virol. 55, 206-212; Rice et al., (1985) J. Virol. 56:767-778).

[0084] We therefore generate packaging cell lines of monkey origin (e.g., VERO, CV1) that harbor:

[0085] a) E1 sequences, to allow replication of E1/E2 defective adenoviruses; and

[0086] b) E2A sequences, containing the hr mutation and the ts125 mutation, named ts400 (Brough et al, (1985) J. Virol. 55:206-212; Rice et al., (1985) J. Virol. 56:767-778 to prevent cell death by E2A overexpression; and/or

[0087] c) E2A sequences, just containing the hr mutation, under the control of an inducible promoter; and/or

[0088] d) E2A sequences, containing the hr mutation and the ts125 mutation (ts400), under the control of an inducible promoter.

[0089] Furthermore, we disclose the construction of novel and improved combinations of (novel and improved) packaging cell lines and (novel and improved) recombinant adenoviral vectors.

[0090] We provide:

[0091] 1) A novel packaging cell line derived from diploid HER cells that harbors nucleotides 80-5788 of the Ad5 genome. This cell line, named 911, (deposited under ECACC deposit accession number 95062101 under the provision of the Budapest Treaty with the Centre for Applied Microbiology and Research Authority (European Collection of Animal Cell Cultures or “ECACC”, Porton Down, Salisbury, Wiltshire SP4, OJG, United Kingdom and International Depository Authority), has many characteristics that make it superior to the commonly used 293 cells (Fallaux et al., (1996) Hum. Gene Ther. 7:215-222).

[0092] 2) Novel packaging cell lines that express just E1A genes and not E1B genes. Established cell lines (and not human diploid cells of which 293 and 911 cells are derived) are able to express E1A to high levels without undergoing apoptotic cell death, as occurs in human diploid cells that express E1A in the absence of E1B. Such cell lines are able to transcomplement E1B-defective recombinant adenoviruses, because viruses mutated for E1B 21 kD protein are able to complete viral replication even faster than wild-type adenoviruses (Telling et al., (1994) J. Virol 68:541-7). The constructs are described in detail below, and graphically represented in FIGS. 1-5. The constructs are transfected into the different established cell lines and are selected for high expression of E1A. This is done by operatively linking a selectable marker gene (e.g NEO gene) directly to the E1B promoter. The E1B promoter is transcriptionally activated by the E1A gene product and therefore resistance to the selective agent (e.g. G418 in the case NEO is used as the selection marker) results in direct selection for desired expression of the E1A gene.

[0093] 3) Packaging constructs that are mutated or deleted for EIB 21 kD, but just express the 55 kD protein.

[0094] 4) Packaging constructs to be used for generating complementing packaging cell lines from diploid cells (not exclusively of human origin) without the need of selection with marker genes. These cells are immortalized by expression of E1A. However, in this particular case expression of E1B is essential to prevent apoptosis induced by E1A proteins. Selection of E1 expressing cells is achieved by selection for focus formation (immortalization), as described for 293 cells (Graham et al.,) J. Gen. Virol. 36:59-72) and 911 cells (Fallaux et al., (1996) Hum. Gene Ther. 7:215-222), that are E1 transformed human embryonic kidney (HEK) cells and HER cells, respectively.

[0095] 5) After transfection of HER cells with construct pIG.E1A.E1B (FIG. 4), seven independent cell lines could be established. These cell lines were designated PER.C1, PER.C3, PER.C4, PER.C5, PER.C6, PER.C8 and PER.C9. PER denotes PGK-E1-Retinoblasts. These cell lines express E1A and E1B proteins, are stable (e.g. PER.C6 for more than 57 passages) and complement E1-defective adenoviral vectors. Yields of recombinant adenovirus obtained on PER cells are a little higher than obtained on 293 cells. One of these cell lines (PER.C6) has been deposited at the ECACC under number 96022940.

[0096] 6) New adenoviral vectors with extended E1 deletions (deletion nt. 459-3510). Those viral vectors lack sequences homologous to E1 sequences in said packaging cell lines. These adenoviral vectors contain pIX promoter sequences and the pIX gene, as pIX (from its natural promoter sequences) can only be expressed from the vector and not by packaging cells (Matsui et al., (1986) Mol. Cell Biol. 6:4149-4154, Hoeben and Fallaux, pers. comm.; Imler et al., (1996) Gene Ther. 3:75-84).

[0097] 7) E2A expressing packaging cell lines preferably based on either E1A expressing established cell lines or E1A+E1B expressing diploid cells (see, under 2-4). E2A expression is either under the control of an inducible promoter or the E2A ts125 mutant is driven by either an inducible or a constitutive promoter.

[0098] 8) Recombinant adenoviral vectors as described before (see, 6) but carrying an additional deletion of E2A sequences.

[0099] 9) Adenovirus packaging cells from monkey origin that are able to transcomplement E1-defective recombinant adenoviruses. They are preferably co-transfected with pIG.E1AE1B and pIG.NEO, and selected for NEO resistance. Such cells expressing E1A and E1B are able to transcomplement E1 defective recombinant human adenoviruses, but will do so inefficiently because of a block of the synthesis of late adenoviral proteins in cells of monkey origin (Klessig et al., (1979) Cell 17:957-966). To overcome this problem, we generate recombinant adenoviruses that harbor a host-range mutation in the E2A gene, allowing human adenoviruses to replicate monkey cells. Such viruses are generated as described in FIG. 12, except DNA from a hr-mutant is used for homologous recombination.

[0100] 10) Adenovirus packaging cells from monkey origin as described under 9, except that they will also be co-transfected with E2A sequences harboring the hr mutation. This allows replication of human adenoviruses lacking E1 and E2A (see, under 8). E2A in these cell lines is either under the control of an inducible promoter or the tsE2A mutant is used. In the latter case, the E2A gene will thus carry both the ts mutation and the hr mutation (derived from ts400). Replication competent human adenoviruses have been described that harbor both mutations (Brough et al., J. Virol. 55:206-212; Rice et al., (1985) J. Virol. 56:767-778).

[0101] Furthermore, the present invention, in one aspect, provides new cosmid and plasmid vectors containing large fragments of the adenoviral genome and an improved method for generating recombinant adenoviral vectors by making use of these cloned adenovirus sequences.

[0102] Accordingly, the present invention provides a new system of generating recombinant adenoviruses that is fast, highly flexible, reliable and only requires standard cloning technology. The new system is surprisingly efficient in generating recombinant adenoviruses. In combination with the packaging cells of the invention, it ensures RCA-free generation and propagation of recombinant adenoviruses. The previously identified problems associated with current methods to generate recombinant adenoviruses are, in one aspect, solved by using a functional combination of cloned adenovirus sequences and an intra-cellular homologous recombination in suitable packaging cells.

[0103] Accordingly, the present invention provides methods and means to efficiently generate and produce vectors that are able to harbor very large fragments of (genomic) DNA. Vectors of the invention can be safely produced to very high titers and are able to transduce mammalian cells, including human cells, with high efficiency, thereby favoring homologous recombination with (genomic) DNA molecules present in the mammalian cells, due to the high numbers of introduced DNA molecules and their large homologous overlap with the target DNA molecules for recombination. In one aspect, vectors according to the invention are based on adenoviral vectors derived from an adenoviral genome, from which as much as possible of the adenoviral genome is deleted except for the ITR sequences and the sequences needed in cis for packaging (minimal adenoviral vectors). Such vectors can accommodate up to 38 kb of foreign (genomic) DNA.

[0104] Minimal adenoviral vectors with large genomic sequences functioning as gene replacement vectors can be generated efficiently using the plasmid-based intracellular PCR system disclosed infra, thereby avoiding the need of contaminating helper viruses. In addition, we disclose an alternative way of producing minimal adenoviral vectors without the need for helper viruses. Replication and packaging of the minimal adenoviral vectors with large inserts can also be achieved by using them in combination with a complementing molecule containing all parts of the adenoviral genome that are required for replication and packaging except for the packaging signal and E1 sequences. Such a complementing molecule need not necessarily replicate by the virtue of the adenoviral replication machinery. It may, for example, be cloned on a plasmid that also contains the SV40 origin of replication. Transfection of this DNA together with the minimal adenoviral vector in a E1-containing packaging cell that also (inducibly) expresses the SV40 Large T protein will lead to replication of the adenovirus molecule and expression of adenoviral proteins. The latter will then initiate replication and packaging of the minimal adenoviral vectors.

[0105] A further aspect of the invention provides otherwise improved adenoviral vectors, as well as novel strategies for generating and applying such vectors as well as a method for the intracellular amplification of linear DNA fragments in mammalian cells.

[0106] The so-called “minimal” adenoviral vectors according to the present invention retain at least a portion of the viral genome that is required for encapsulation of the genome into virus particles (the encapsulation signal), as well as at least one copy of at least a functional part or a derivative of the Inverted Terminal Repeat (ITR), that is DNA sequences derived from the termini of the linear adenovirus genome. The vectors, according to the present invention will also contain a transgene linked to a promoter sequence to govern expression of the transgene. Packaging of the so-called minimal adenoviral vector can be achieved by co-infection with a helper virus or, alternatively, with a packaging deficient replicating helper system as described hereinafter.

[0107] Adenovirus-derived DNA fragments that can replicate in suitable cell lines and that may serve as a packaging deficient replicating helper system are generated as herein described. These DNA fragments retain at least a portion of the transcribed region of the “late” transcription unit of the adenovirus genome and carry deletions in at least a portion of the E1 region and deletions in at least a portion of the encapsulation signal. In addition, these DNA fragments contain at least one copy of an ITR. An ITR is located at one terminus of the transfected DNA molecule. The other end may contain an ITR, or alternatively, a DNA sequence that is complementary to a portion of the same strand of the DNA molecule other than the ITR. If, in the latter case, the two complementary sequences anneal, the free 3′-hydroxyl group of the 3′ terminal nucleotide of the hairpin structure can serve as a primer for DNA synthesis by cellular and/or adenovirus-encoded DNA polymerases, resulting in conversion into a double-stranded form of at least a portion of the DNA molecule. Further replication initiating at the ITR will result in a linear double-stranded DNA molecule, that is flanked by two ITR's, and is larger than the original transfected DNA molecule (see, FIG. 13). This molecule can replicate itself in the transfected cell by virtue of the adenoviral proteins encoded by the DNA molecule and the adenoviral and cellular proteins encoded by genes in the host cell genome. This DNA molecule cannot be encapsulated due to its large size (greater than 39,000 bp) or due to the absence of a functional encapsulation signal. This DNA molecule is intended to serve as a helper for the production of defective adenoviral vectors in suitable cell lines.

[0108] The invention also includes a method for amplifying linear DNA fragments of variable size in suitable mammalian cells. These DNA fragments contain at least one copy of the ITR at one terminus of the fragment. As described above, the other end may contain an ITR, or alternatively, a DNA sequence that is complementary to a portion of the same strand of the DNA molecule other than the ITR. If, in the latter case, the two complementary sequences anneal, the free 3′-hydroxyl group of the 3′ terminal nucleotide of the hairpin-structure can serve as a primer for DNA synthesis by cellular and/or adenovirus-encoded DNA polymerases, resulting in conversion of the displaced strand into a double stranded form of at least a portion of the DNA molecule. Further replication initiation at the ITR will result in a linear double-stranded DNA molecule, that is flanked by two ITR's, which is larger than the original transfected DNA molecule. A DNA molecule that contains ITR sequences at both ends can replicate itself in transfected cells by virtue of the presence of at least the adenovirus E2 proteins (namely the DNA-binding protein DBP, the adenovirus DNA polymerase (Ad-pol), and the pre-terminal protein (pTP). The required proteins may be expressed from adenoviral genes on the DNA molecule itself, from adenoviral E2 genes integrated in the host-cell genome, or from a replicating helper fragment as described above.

[0109] Several groups have shown that the presence of ITR sequences at the end of DNA molecules are sufficient to generate adenovirus mini-chromosomes that can replicate, if the adenovirus-proteins required for replication are provided in trans, for example, by infection with a helper virus (Hu et al., (1992) Gene 110:145-150); (Wang et al., (1985) in vivo. Nucl. Acids Res. 13:5173-5187); Hay et al., (1984) J. Mol. Biol. 174:493-510). Hu et al., (1992) Gene 110:145-150, observed the presence and replication of symmetrical adenovirus minichromosome-dimers after transfection of plasmids containing a single ITR. The authors were able to demonstrate that these dimeric mini-chromosomes arise after tail-to-tail ligation of the single ITR DNA molecules. In DNA extracted from defective adenovirus type 2 particles, dimeric molecules of various sizes have also been observed using electron-microscopy (Daniell J. Virol. 19:685-708 (1976)). It was suggested that the incomplete genomes were formed by illegitimate recombination between different molecules and that variations in the position of the sequence at which the illegitimate base pairing occurred were responsible for the heterogeneous nature of the incomplete genomes. Based on this mechanism, it was speculated that, in theory, defective molecules with a total length of up to two times the normal genome could be generated. Such molecules could contain duplicated sequences from either end of the genome. However, no DNA molecules larger than the full-length virus were found packaged in the defective particles (Daniell J. Virol. 19:685-708 (1976)). This can be explained by the size-limitations that apply to the packaging. In addition, it was observed that, in the virus particles, DNA-molecules with a duplicated left-end predominated over those containing the right-end terminus. This is fully explained by the presence of the encapsulation signal near that left-end of the genome (Grable et al., (1990) J. Virol. 64:2047-2056; Grable et al., (1992) J. Virol. 66:723-731; Hearing et al., (1987) J. Virol. 61:2555-2558).

[0110] The major problems associated with the current adenovirus-derived vectors are:

[0111] a) The strong immunogenicity of the virus particle.

[0112] b) The expression of adenoviral genes that reside in the adenoviral vectors, resulting in a Cytotoxic T-cell response against the transduced cells.

[0113] c) The low amount of heterologous sequences that can be accommodated in the current vectors (up to maximally approximately 8,000 bp. of heterologous DNA).

[0114] Ad A) The strong immunogenicity of the adenovirus particle results in a host immunological response, even after a single administration of the adenoviral vector. As a result of the development of neutralizing antibodies, a subsequent administration of the virus will be less effective or even completely ineffective. However, a prolonged or persistent expression of the transferred genes will reduce the number of administrations required and may bypass the problem.

[0115] Ad B) Experiments performed by Wilson and collaborators have demonstrated that after adenovirus-mediated gene transfer into immunocompetent animals, the expression of the transgene gradually decreases and disappears approximately 2-4 weeks post-infection (Yang et al., (1994a) Proc Natl Acad Sci USA 91:4407-11; Yang et al., (1994b) Nat Genet. 7:362-369). This is caused by the development of a cytotoxic T-cell (“CTL”) response against the transduced cells. The CTLs were directed against adenoviral proteins expressed by the viral vectors. In the transduced cells, synthesis of the adenovirus DNA-binding protein (the E2A-gene product), penton and fiber proteins (late-gene products) could be established. These adenoviral proteins, encoded by the viral vector, were expressed despite deletion of the E1 region. This demonstrates that deletion of the E1 region is not sufficient to completely prevent expression of the viral genes (Engelhardt et al., (1994a) Human Gene Ther. 5:1217-1229).

[0116] Ad C) Studies by Graham and collaborators have demonstrated that adenoviruses are capable of encapsulating DNA of up to 105% of the normal genome size (Bett et al., (1993) J. Virol. 67:5911-5921). Larger genomes tend to be unstable resulting in loss of DNA sequences during propagation of the virus. Combining deletions in the E1 and E3 regions of the viral genomes increases the maximum size of the foreign DNA that can be encapsulated to approx. 8.3 kb. In addition, some sequences of the E4 region appear to be dispensable for virus growth (adding another 1.8 kb to the maximum encapsulation capacity). Also the E2A region can be deleted from the vector, when the E2A gene product is provided in trans in the encapsulation cell line, adding another 1.6 kb. It is, however, unlikely that the maximum capacity of foreign DNA can be significantly increased further than 12 kb.

[0117] We developed a new strategy for generating and producing helper-free stocks of recombinant adenoviral vectors that can accommodate up to 38 kb of foreign DNA. Only two functional ITR sequences and sequences that can function as an encapsulation signal, need to be part of the vector genome. Such vectors are called minimal adenovectors. The helper functions for the minimal adenovectors are provided in trans by encapsulation defective-replication competent DNA molecules that contain all the viral genes encoding the required gene products, with the exception of those genes that are present in the host-cell genome, or genes that reside in the vector genome.

[0118] The applications of the disclosed inventions are outlined below and are illustrated in the Examples.

[0119] Use of the IG Packaging Constructs Diploid Cells.

[0120] The constructs, in particular pIG.E1A.E1B, will be used to transfect diploid human cells, such as HER cells, HEK cells, and Human Embryonic Lung (“HEL”) cells. Transfected cells will be selected for transformed phenotype (focus formation) and tested for their ability to support propagation of E1-deleted recombinant adenovirus, such as IG.Ad.MLPI.TK. Such cell lines will be used for generating and (large-scale) producing E1-deleted recombinant adenoviruses. Such cells, infected with recombinant adenovirus are also intended to be used in vivo as a local producer of recombinant adenovirus, for example, for the treatment of solid tumors. 911 cells are used for the titration, generation and production of recombinant adenoviral vectors (Fallaux et al., (1996) Hum. Gene Ther. 7:215-222).

[0121] HER cells transfected with pIG.E1A.E1B have resulted in 7 independent clones (called PER cells). These clones are used for the production of E1 deleted (including non-overlapping adenoviral vectors) or E1 defective recombinant adenoviral vectors and provide the basis for introduction of, for example, E2B or E2A constructs (e.g., ts125E2A, see, below), E4 etc., that will allow propagation of adenoviral vectors that have mutations in, for example, E2A or E4. In addition, diploid cells of other species that are permissive for human adenovirus, such as the cotton rat (Sigmodon hispidus) (Pacini et al., (1984) J. Infect. Dis. 150:92-97), Syrian hamster (Morin et al., (1987) Proc. Natl. Acad. Sci. USA 84:4626-4630) or chimpanzee (Levrero et al, (1991) Gene 101:195-202), will be immortalized with these constructs. Such cells, infected with recombinant adenovirus, are also intended to be used in vivo for the local production of recombinant adenovirus, for example, for the treatment of solid tumors.

[0122] Established Cells.

[0123] The constructs, in particular pIG.E1A.NEO, can be used to transfect established cells, for example, A549 (human bronchial carcinoma), KB (oral carcinoma), MRC-5 (human diploid lung cell line) or GLC cell lines (small cell lung cancer) de Leij et al., (1985) Cancer Res. 45:6024-6033; Postmus et al., (1988) Eur. J. Clin. Oncol. 24:753-763) and selected for NEO resistance. Individual colonies of resistant cells are isolated and tested for their capacity to support propagation of E1-deleted recombinant adenovirus, such as IG.Ad.MLPI.TK. When propagation of E1 deleted viruses on E1A containing cells is possible, such cells can be used for the generation and production of E1-deleted recombinant adenovirus. They are also used for the propagation of E1A deleted/E1B retained recombinant adenovirus.

[0124] Established cells can also be co-transfected with pIG.E1A.E1B and pIG.NEO (or another NEO containing expression vector). Clones resistant to G418 are tested for their ability to support propagation of E1-deleted recombinant adenovirus, such as IG.Ad.MLPI.TK and used for generating and producing E1-deleted recombinant adenovirus and will be applied in vivo for local production of recombinant virus, as described for the diploid cells (see, above).

[0125] All cell lines, including transformed diploid cell lines orNEO-resistant established lines, can be used as the basis for generating “next generation” packaging cell lines, that support propagation of E1-defective recombinant adenoviruses, that also carry deletions in other genes, such as E2A and E4. Moreover, they will provide the basis for the generation of minimal adenoviral vectors as disclosed herein.

[0126] E2 expressing Cell Lines

[0127] Packaging cells expressing E2A sequences are and will be used for generating and (large scale) producing E2A-deleted recombinant adenovirus. The newly generated human adenovirus packaging cell lines or cell lines derived from species permissive for human adenovirus (E2A or ts125E2A; E1A+E2A; E1A+E1B+E2A; E1A+E2A/ts125; E1A+E1B+E2A/ts125) or non-permissive cell lines such as monkey cells (hrE2A or hr+ts125E2A; E1A+hrE2A; E1A+E1B+hrE2A; E1A+hrE2A/ts125; E1A+E1B+hrE2A/ts125) are and will be used for generating and (large scale) producing E2A deleted recombinant adenoviral vectors. In addition, they will be applied in vivo for local production of recombinant virus, as described for the diploid cells (see, above).

[0128] Novel Adenoviral Vectors

[0129] The newly developed adenoviral vectors harboring an E1 deletion of nucleotides 459-3510 will be used for gene transfer purposes. These vectors are also the basis for the development of further deleted adenoviral vectors that are mutated for, for example, E2A, E2B or E4. Such vectors will be generated, for example, on the newly developed packaging cell lines described herein (see, 1-3 above).

[0130] Minimal Adenovirus Packaging System

[0131] We disclose adenovirus packaging constructs (to be used for the packaging of minimal adenoviral vectors) may have the following characteristics:

[0132] a) The packaging construct replicates.

[0133] b) The packaging construct cannot be packaged because the packaging signal is deleted.

[0134] c) The packaging construct contains an internal hairpin-forming sequence (see, section “Experimental; suggested hairpin” see, FIG. 15)

[0135] d) Because of the internal hairpin structure, the packaging construct is duplicated, that is, the DNA of the packaging construct becomes twice as long as it was before transfection into the packaging cell (in our sample it duplicates from 35 kb to 70 kb). This duplication also prevents packaging. Note that this duplicated DNA molecule has ITR's at both termini (see, e.g., FIG. 13)

[0136] e) This duplicated packaging molecule is able to replicate like a “normal adenovirus” DNA molecule

[0137] f) The duplication of the genome is a prerequisite for the production of sufficient levels of adenoviral proteins, required to package the minimal adenoviral vector

[0138] g) The packaging construct has no overlapping sequences with the minimal vector or cellular sequences that may lead to generation of RCA by homologous recombination.

[0139] This packaging system will be used to produce minimal adenoviral vectors. The advantages of minimal adenoviral vectors, for example, for gene therapy of vaccination purposes, are well known (accommodation of up to 38 kb; “gutting” of all potentially toxic and immunogenic adenoviral genes).

[0140] Adenoviral vectors containing mutations in essential genes (including minimal adenoviral vectors) can also be propagated using this system.

[0141] Use of Intracellular E2-expressing Vectors

[0142] Minimal adenoviral vectors are generated using the helper flnctions provided in trans by packaging-deficient replicating helper molecules. The adenovirus-derived ITR sequences serve as origins of DNA replication in the presence of at least the E2-gene products. When the E2 gene products are expressed from genes in the vector genome (N.B. the gene(s) must be driven by an E1-independent promoter), the vector genome can replicate in the target cells. This will allow a significantly increased number of template molecules in the target cells, and, as a result an increased expression of the genes of interest encoded by the vector. This is of particular interest for approaches of gene therapy in cancer.

[0143] Applications of Intracellular Amplification of Linear DNA Fragments.

[0144] A similar approach could also be taken if amplification of linear DNA fragments is desired. DNA fragments of known or unknown sequence could be amplified in cells containing the E2-gene products if at least one ITR sequence is located near or at its terminus. No apparent constraints exist on the size of the fragment. Even fragments much larger than the adenovirus genome (36 kb) could be amplified using this approach. It is thus possible to clone large fragments in mammalian cells without either shuttling the fragment into bacteria (such as E. coli) or using the polymerase chain reaction (“PCR”). At the end stage of a productive adenoviral infection, a single cell can contain over 100,000 copies of the viral genome. In the optimal situation, the linear DNA fragments can be amplified to similar levels. Thus, one should be able to extract more than 5 μg of DNA fragment per 10 million cells (for a 35-kbp fragment). This system can be used to express heterologous proteins (equivalent to the Simian Virus 40-based COS-cell system) for research or for therapeutic purposes. In addition, the system can be used to identify genes in large fragments of DNA. Random DNA fragments may be amplified (after addition of ITRs) and expressed during intracellular amplification. Election or, selection of those cells with the desired phenotype can be used to enrich the fragment of interest and to isolate the gene.

[0145] Gene Correction Vectors.

[0146] Gene therapy procedures may be divided into two different concepts, i.e., gene addition and gene replacement. Gene addition aims at introducing a therapeutic nucleic acid molecule into somatic cells of a patient, whereby expression of said therapeutic nucleic acid molecule is often under the control of a heterologous promoter and transcription termination signal. For example, when a patient suffers from an inherited disease, a functional copy of the defective nucleic acid molecule responsible for the disease phenotype is introduced into cells of the patient and, upon expression of the therapeutic nucleic acid molecule, the disease phenotype is corrected. Gene addition may also be used to accomplish expression of otherwise not expressed genes, such as, for example, cytokine or suicide genes like Herpes Simplex Virus Thymidine Kinase (“HSV-TK”) to treat tumors. The gene replacement procedure aims at repairing at least one copy of a defective gene responsible for a disease phenotype. This can be achieved by introducing a functional version of a gene, or part thereof including the mutant site of that gene, in such a way that homologous recombination between the functional version and said defective gene occurs. Consequently, the defective gene or its mutant site is replaced by the functional version of that gene or part thereof. In this way, no nucleic acid material that is foreign to the species of which the patient is a member is expressed in the treated cells but at least one allele of the mutant gene is repaired. For the majority of the inherited diseases, it is known that heterozygous carriers are not affected, or at least are affected to a lesser extent than a homozygous patient. Thus, gene replacement may be used for correction of inherited disorders. It is to be understood that this also includes the repair of defective tumor suppressor genes.

[0147] For gene therapy purposes, it is preferable to retain the E3 region. E3-containing vectors will be superior to their E3-deleted counterparts because they are able to prevent or reduce host cell responses such as CTL lysis of adenovirus infected cells and cell lysis by TNF.

[0148] It will be understood that it may not be necessary to retain the whole E3 region in the vectors according to the invention, as long as the part retained still has the function of reducing the response of the host against infected cells. For example, expression of E3-14.7 kD alone may be sufficient to reduce early responses mediated by TNF (see, Ginsberg, H. S. (1989) Proc. Natl. Acad. Sci, USA 86:3823-3827; Ginsberg, H. S. (1991) Proc. Natl. Acad. Sci. USA 88:1651-1655). These vectors are useful for gene therapy of inherited diseases such as cystic fibrosis, Duchenne molecular dystrophy, Hypercholesterolemia, blood clotting disorders (hemophilia) and the like. They also are useful in the therapy of acquired diseases, such as tumors, hepatitis, (auto) immune diseases, restenosis, rheumatoid and the like.

[0149] Advantages of gene replacement over gene addition include (1) expression regulation of the replacing gene is identical to the endogenous expression pattern, and (2) the procedure is safe, because no risk exists of insertion mutagenesis due to random integration.

[0150] The following examples are presented as illustrations, not limitations.

EXAMPLES Example 1

[0151] Generation of Cell Lines Able to Transcomplement E1 Defective Recombinant Adenoviral Vectors

[0152] 1. 911 Cell Line

[0153] A cell line that harbors E1 sequences of Ad5, able to trans-complement E1-deleted recombinant adenovirus has been generated (Fallaux et al., Hum. Gene Ther. 7:215-222 (1996)). This cell line was obtained by transfecting human diploid HER cells with pAd5XhoIC, that contains nt 80-5788 of Ad5; one of the resulting transformants was designated 911. This cell line has been shown to be useful in the propagation of E1 defective recombinant adenovirus. It was found to be superior to the 293 cells. Unlike 293 cells, 911 cells lack a fully transformed phenotype, which most likely is the cause of performing better as adenovirus packaging line. For example, it was found that plaque assays can be performed faster (4-5 days instead of 8-14 days on 293); monolayers of 911 cells survive better under agar overlay as required for plaque assays; and there is a higher amplification of E1-deleted vectors. In addition, unlike 293 cells that were transfected with sheared adenoviral DNA, 911 cells were transfected using a defined construct. Transfection efficiencies of 911 cells are comparable to those of 293.

[0154] New Packaging Constructs

[0155] Source of Adenovirus Sequences

[0156] Adenovirus sequences are derived either from pAd5.SalB, containing nt 80-9460 of human Ad5 (Bernards et al., (1983) Virology 127:45-53) or from wild-type Ad5 DNA. PAd5.SalB was digested with Sal I and Xho I and the large fragment was religated and this new clone was named pAd5.X/S. The pTN construct (constructed by Dr. R. Vogels, IntroGene, The Netherlands) was used as a source for the human PGK promoter and the NEO gene.

[0157] Human PGK Promoter and NEO^(R) Gene

[0158] Transcription of E1A sequences in the new packaging constructs is driven by the human PGK promoter (Michelson et al., (1983) Proc. Natl. Acad. Sci. USA 80:472-476); Singer-Sam et al., (1984) Gene 32:409-417), derived from plasmid pTN (gift of R. Vogels), which uses pUC119 (Vieira et al., (1987) pp. 3-11: Methods in Enzymology, Acad. Press Inc.) as a backbone. This plasmid was also used as a source for the NEO gene fused to the Hepatitis B Virus (“HBV”) poly-adenylation signal.

[0159] Fusion of PGK Promoter to E1 Genes (FIG. 1)

[0160] In order to replace the E1 sequences of Ad5 (ITR, origin of replication and packaging signal) by heterologous sequences, we have amplified E1 sequences (nt 459 to nt 960) of Ad5 by PCR, using primers Ea1 and Ea2 (see, Table I). The resulting PCR product was digested with ClaI and ligated into Bluescript (Stratagene), predigested with Cla I and EcoR V, results in construct pBS.PCRI.

[0161] Vector pTN was digested with restriction enzymes EcoR I (partially) and Sca I, and the DNA fragment containing the PGK promoter sequences was ligated into PBS.PCRI digested with Sca I and EcoR I. The resulting construct PBS.PGK.PCRI contains the human PGK promoter operatively linked to Ad5 E1 sequences from nt.459 to nt.916.

[0162] Construction of pIG.E1A.E1B (FIG. 2)

[0163] PIG.E1A.E1B.X contains the E1A and E1B coding sequences under the direction of the PGK promoter. As Ad5 sequences from nt.459 to nt.5788 are present in this construct, also pIX protein of adenovirus is encoded by this plasmid. pIG.E1A.E1B.X was made by replacing the Sca I-BspE I fragment ofpAT-X/S by the corresponding fragment from PBS.PGK.PCRI (containing the PGK promoter linked to E1A sequences).

[0164] Construction of PIG.E1A.NEO (FIGS. 3A and 3B)

[0165] In order to introduce the complete E1B promoter and to fuse this promoter in such a way that the AUG codon of E1B 21 kD exactly functions as the AUG codon of NEO^(R), the E1B promoter was amplified using primers Ea3 and Ep2, where primer Ep2 introduces a Nco I site in the PCR fragment. The resulting PCR fragment, named PCRII, was digested with Hpa I and Nco I and ligated into pAT-X/S, which was predigested with Hpa I and with NcoI. The resulting plasmid was designated pAT-X/S-PCR2. The Nco I-Stu I fragment of pTN, containing the NEO gene and part of the HBV poly-adenylation signal, was cloned into pAT-X/S-PCR2 which had been digested with Nco I and Nru I. The resulting construct was pAT-PCR2-NEO. The poly-adenylation signal was completed by replacing the Sca I-Sal I fragment of pAT-PCR2.NEO with the corresponding fragment of pTN, resulting in pAT.PCR2.NEO.p (A). The Sca I-Xba I of pAT.PCR2.NEO.p (A) was replaced with the corresponding fragment of pIG.E1A.E1B-X, containing the PGK promoter linked to E1A genes. The resulting construct was named pIG.E1A.NEO, and thus contains Ad5 E1 sequences (nt.459 to nt.1713) under the control of the human PGK promoter.

[0166] Construction of pIG.E1A.E1B (FIG. 4)

[0167] pIG.E1A.E1B contains nt.459 to nt.3510 of Ad5, that encode the E1A and E1B proteins. The E1B sequences are terminated at the splice acceptor at nt.3511. No pIX sequences are present in this construct.

[0168] pIG.E1A.E1B was made as follows: The sequences encoding the N-terminal amino acids of E1B 55kd were amplified using primers Eb1 and Eb2 which introduces a Xho I site. The resulting PCR fragment was digested with Bgl II and cloned into Bl II/Nru I of pAT-X/S, thereby obtaining pAT-PCR3. The HBV poly (A) sequences of pIG.E1A.NEO were introduced downstream of the EIB sequences of pAT-PCR3 by exchange of the Xba-Sal I fragment of pIG.E1A.NEO and the Xba I Xho I fragment of pAT.PCR3.

[0169] Construction of pIG.NEO (FIG. 5)

[0170] This construct is of use when established cells are transfected with E1A.E1B constructs and NEO selection is required. Because NEO expression is directed by the E1B promoter, NEO resistant cells are expected to co-express. E1A, which also is advantageous for maintaining high levels of expression of E1A during long-term culture of the cells. pIG.NEO was generated by cloning the Hpa I-Sca I fragment of pIG.E1A.NEO, containing the NEO gene under the control of the Ad5 EIB promoter, into pBS digested with EcoR V and Sca I.

[0171] Testing of Constructs

[0172] The integrity of the constructs pIG.E1A.NEO, pIG.E1A.E1B.X and pIG.E1A.E1B was assessed by restriction enzyme mapping. Furthermore, parts of the constructs that were obtained by PCR analysis were confirmed by sequence analysis. No changes in the nucleotide sequence were found.

[0173] The constructs were transfected into primary BRK (Baby Rat Kidney) cells and tested for their ability to immortalize (pIG.E1A.NEO) or fully transform (pAd5.XhoIC,pIG.E1A.E1B.X and pIG.E1A.E1B) these cells. Kidneys of 6-day old WAG-Rij rats were isolated, homogenized and trypsinized. Subconfluent dishes (diameter 5 cm) of the BRK cell cultures were transfected with 1 or 5 μg of pIG.NEO, pIG.E1A.NEO, pIG.E1A.E1B, pIG/E1A.E1B.X, pAd5XhiIC, or with pIG.E1A.NEO together with PDC26 (Elsen et al., (1983) Virology 128:377-390), carrying the Ad5.E1B gene under control of the SV40 early promoter. Three weeks post-transfection, when foci were visible, the dishes were fixed, Giemsa stained and the foci counted.

[0174] An overview of the generated adenovirus packaging constructs, and their ability to transform BRK, is presented in FIG. 6. The results indicate that the constructs pIG.E1A.E1B and pIG.E1A.E1B.X are able to transform BRK cells in a dose-dependent manner. The efficiency of transformation is similar for both constructs and is comparable to what was found with the construct that was used to make 911 cells, namely pAd5.XhoIC.

[0175] As expected, pIG.E1A.NEO was hardly able to immortalize BRK. However, co-transfection of an E1B expression construct (PDC26) did result in a significant increase of the number of transformants (18 versus 1), indicating that the E1A encoded by pIG.E1A.NEO is functional. We conclude therefore, that the newly generated packaging constructs are suitable for the generation of new adenovirus packaging lines.

[0176] Generation of Cell Lines with New Packaging Constructs Cell Lines and Cell Culture.

[0177] Human A549 bronchial carcinoma cells (Shapiro et al., (1978) Biochem. Biophys. Acta 530:197-207), HER cells, Ad5-E1-transformed HEK cells (293; Graham et al., (1977) J. Gen. Virol. 36:59-72) and Ad5-transformed HER cells (911; Fallaux et al., (1996). Hum. Gene Ther. 7:215-222) and PER cells were grown in Dulbecco's Modified Eagle Medium (“DMEM”) supplemented with 10% Fetal Calf Serum (FCS) and antibiotics in a 5% CO₂ atmosphere at 37° C. Cell culture media, reagents and sera were purchased from Gibco Laboratories (Grand Island, N.Y.). Culture plastics were purchased from Greiner (Nurtingen, Germany) and Coming (Corning, N.Y.).

[0178] Viruses and Virus Techniques

[0179] The construction of recombinant adenoviral vectors IG.Ad.MLP.nls.lacZ, IG.Ad.MLP.luc, IG.Ad.MLP.TK and IG.Ad.CMV.TK is described in detail in patent application EPO Patent Application EP 95202213. The recombinant adenoviral vector IG.Ad.MLP.nls.lacZ contains the E. coli lacZ gene, encoding β-galactosidase, under control of the Ad2 major late promoter (MLP), IG.Ad.MLP.luc contains the firefly luciferase gene drive by the Ad2 MLP, and adenoviral vectors IG.Ad.MLP.TK and IG.Ad.CMV.TK contain the HSV-TK gene under the control of the Ad2 MLP and the Cytomegalovirus (CMV) enhancer/promoter, respectively.

[0180] Transfections

[0181] All transfections were performed by calcium-phosphate precipitation DNA (Graham et al., (1973) Virology 52:456-467) with the GIBCO Calcium Phosphate Transfection System (GIBCO BRL Life Technologies, Inc., Gaithersburg, USA), according to the manufacturer's protocol.

[0182] Western Blotting

[0183] Subconfluent cultures of exponentially growing 293,911 and Ad5-E 1-transformed A549 and PER cells were washed with PBS and scraped in Fos-RIPA buffer (10 mM Tris (pH 7,5), 150 mMNaCl, 1% NP40,01% sodium dodecyl sulfate (SDS), 1% NA-DOC, 0,5 mM phenyl methyl sulfonyl fluoride (PMSF), 0,5 mM trypsin inhibitor, 50 mM NaF and 1 mM sodium vanadate). After 10 min. at room temperature, lysates were cleared by centrifugation. Protein concentrations were measured with the BioRad protein assay kit, and 25 μg total cellular protein was loaded on a 12.5% SDS-PAA gel. After electrophoresis, proteins were transferred to nitrocellulose (1 h at 300 mA). Prestained standards (Sigma, USA) were run in parallel. Filters were blocked with 1% bovine serum albumin (BSA) in TBST (10 mM Tris, pH 8, 15 mM NaCl, and 0.05% Tween-20) for 1 hour. First antibodies were the mouse monoclonal anti-Ad5-E1B-55-kDa antibody A1C6 (Zantema et al., unpublished), the rat monoclonal anti-Ad5-E1B-221-kDa antibody C1G111 (Zantema et al., (1985) Virology 142:44-58). The second antibody was a horseradish peroxidase-labeled goat anti-mouse antibody (Promega). Signals were visualized by enhanced chemoluminescence (Amersham Corp. UK).

[0184] Southern Blot Analysis

[0185] High molecular weight DNA was isolated and 10 μg was digested to completion and fractionated on a 0.7% agarose gel. Southern blot transfer to Hybond N+ (Amersham, UK) was performed with a 0.4 M NAOH, 0.6 M NaCl transfer solution (Church and Gilbert, 1984). Hybridization was performed with a 2463-nt Ssp I-Hind III fragment from pAd5.SalB (Bernards et al., (1983) Virology 127:45-53). This fragment consists of Ad5 bp. 342-2805. The fragment was radio labeled with α⁻³²=dCTP with the use of random hexanucleotide primers and Kelnow DNA polymerase. The southern blots were exposed to a Kodak XAR-5 film at −80° C. and to a Phospho-Imager screen which was analyzed by B&L systems Molecular Dynamics Software.

[0186] A549

[0187] Ad5-E1-transformed A549 human bronchial carcinoma cell lines were generated by transfection with pIG.E1A.NEO and selection for G418 resistance. Thirty-one G418 resistant clones were established. Co-transfection of pIG.E1A.E1B with pIG.NEO yielded seven G418 resistant cell lines.

[0188] PER

[0189] Ad5-E1-transformed HER cells were generated by transfection of primary HER cells with plasmid pIG.E1A.E1B. Transformed cell lines were established from well-separated foci. We were able to establish seven clonal cell lines, which we called PER.C1, PER.C3, PER.C4, PER.C5, PER.C6, PER.C8 and PER.C9. One of the PER clones, namely PER.C6, has been deposited at the ECACC under number 96022940.

[0190] Expression of Ad5 E1A and E1B Genes in Transformed A549 and PER Cells

[0191] Expression of the Ad5 E1A and the 55-kDa and 21 kDa E1B proteins in the established A549 and PER cells was studied by means of Western blotting, with the use of monoclonal antibodies (mAb). mAb M73 recognizes the E1A products, whereas Mabs AIC6 and CIGll are directed against the 55-kDa and 21 kDa E1B proteins, respectively. The antibodies did not recognize proteins in extracts from the parental A549 or the primary HER cells (data not shown). None of the A549 clones that were generated by co-transfection of pIG.NEO and pIG.E1A.E1B expressed detectable levels of E1A or E1B proteins (not shown). Some of the A549 clones that were generated by transfection with pIG.E1A.NEO expressed the Ad5 E1A proteins (FIG. 7), but the levels were much lower than those detected in protein lysates from 293 cells. The steady state E1A levels detected in protein extracts from PER cells were much higher than those detected in extracts from A549-derived cells. All PER cell lines expressed similar levels of E1A proteins (FIG. 7). The expression of the E1B proteins, particularly in the case of E1B 55 kDa, was more variable. Compared to 911 and 293, the majority of the PER clones express high levels of EIB 55 kDa and 2 kDa. The steady state level of EIB 21 kDa was the highest in PER.C3. None of the PER clones lost expression of the Ad5 E1 genes upon serial passage of the cells (not shown). We found that the level of E1 expression in PER cells remained stable for at least 100 population doublings. We decided to characterize the PER clones in more detail.

[0192] Southern Analysis of PER Clones

[0193] To study the arrangement of the Ad5-E1 encoding sequences in the PER clones we performed Southern analysis. Cellular DNA was extracted from all PER clones, and from 293 and 911 cells. The DNA was digested with Hind III, which cuts once in the Ad5 E1 region. Southern hybridization on Hind III-digested DNA, using a radio-labeled Ad5-E1-specific probe revealed the presence of several integrated copies of pIG.E1A.E1B in the genome of the PER clones. FIG. 8 shows the distribution pattern of E1 sequences in the high molecular weight DNA of the different PER cell lines. The copies are concentrated in a single band, which suggests that they are integrated as tandem repeats. In the case of PER.C3, C5, C6 and C9 we found additional hybridizing bands of low molecular weight that indicate the presence of truncated copies of pIG.E1A.E1B. The number of copies was determined with the use of a Phospho-Imager. We estimated that PER.C1, C3, C4, C5, C6, C8 and C9 contain 2, 88, 5, 4, 5, 5, and 3 copies of the Ad5 E1 coding region, respectively, and that 911 and 293 cells contain 1 and 4 copies of the Ad5 E1 sequences, respectively.

[0194] Transfection Efficiency

[0195] Recombinant adenovectors are generated by co-transfection of adaptor plasmids and the large ClaI fragment of Ad5 into 293 cells (EP application 95202213). The recombinant virus DNA is formed by homologous recombination between the homologous viral sequences that are present in the plasmid and the adenovirus DNA. The efficacy of this method, as well as that of alternative strategies, is highly dependent on the transfectability of the helper cells. Therefore, we compared the transfection efficiencies of some of the PER clones with 911 cells, using the E. coli β-galactosidase-encoding lacZ gene as a reporter (FIG. 9).

[0196] Production of Recombinant Adenovirus

[0197] Yields ofrecombinant adenovirus obtained afterinoculationof293,911, PER.C3, PER.C5 and PER.C6 with different adenoviral vectors are presented in Table II.

[0198] The results indicate that the recombinant adenoviral vector yields obtained with PER cells are at least as high as those obtained with the existing cell lines. In addition, the yields of the novel adenoviral vector IG.Ad.MLPI.TK are similar or higher than the yields obtained for the other viral vectors on all cell lines tested.

[0199] Generation of New Adenoviral Vectors (FIG. 10)

[0200] The recombinant adenoviral vectors used (see, EPO Patent Application EP 95202213) are deleted for E1 sequences from 459 to nt. 3328. As construct pE1 A.E1B contains Ad5 sequences nt. 459 to nt.3510 there is a sequence overlap of 183 nt. between E1B sequences in the packaging construct pIG.E1A.E1B and recombinant adenoviruses, such as, for example, IG.Ad.MLP.TK. The overlapping sequences were deleted from the new adenoviral vectors. In addition, non-coding sequences derived from lacZ, that are present in the original constructs, were deleted as well. This was achieved (see, FIG. 10) by PCR amplification of the SV40 poly (A) sequences from pMLP.TK using primers SV40-1 (introduces a BamH I site) and SV40-2 (introduces a Bgl II site). In addition, Ad5 sequences present in this construct were amplified from nt. 2496 (Ad5, introduces a Bgl II site) to nt. 2779 (Ad5-2). Both PCR fragments were digested with Bgl II and were ligated. The ligation product was PCR amplified using primers SV40-1 and Ad5-2. The PCR product obtained was cut with BamH I and Aft II and was ligated into pMLP.TK predigested with the same enzymes. The resulting construct, named pMLPI.TK, contains a deletion in adenoviral E1 sequences from nt.459 to nt. 3510.

[0201] Packaging System

[0202] The combination of the new packaging construct pIG.E1A.E1B and the recombinant adenovirus pMLPI.TK, which do not have any sequence overlap, are presented in FIG. 11. In FIG. 11, the original situation is presented, where the sequence overlap is indicated. The absence of overlapping sequences between pIG.E1A.E1B and pMLPI.TK (FIG. 11a) excludes the possibility of homologous recombination between the packaging construct and the recombinant virus, and is therefore a significant improvement for producing of recombinant adenovirus as compared to the original situation.

[0203] In FIG. 11b the situation is depicted for pIG.E1A.NEO and IG.Ad.MLPI.TK. pIG.E1A.NEO when transfected into established cells, is expected to be sufficient to support propagation of E1-deleted recombinant adenovirus. This combination does not have any sequence overlap, preventing generation of RCA by homologous recombination. In addition, this convenient packaging system allows the propagation of recombinant adenoviruses that are deleted just for E1A sequences and not for E1B sequences.

[0204] Recombinant adenoviruses expressing E1B in the absence of E1A are attractive, as the E1B protein, in particular E1B 19kD, is able to prevent infected human cells from lysis by Tumor Necrosis Factor (TNF) Gooding et al., (1991) J. Virol. 65:3083-3094).

[0205] Generation of Recombinant Adenovirus Derived from pMLPI.TK

[0206] Recombinant adenovirus was generated by co-transfection of 293 cells with Sal I linearized pMLPI.TK DNA and Clal linearized Ad5 wt DNA. The procedure is schematically represented in FIG. 12.

Example 2

[0207] Plasmid-based System for Rapid RCA-free Generation of Recombinant Adenoviral Vectors.

[0208] A. Construction of Adenovirus Clones

[0209] pBr/Ad.Bam-rITR (ECACC Deposit P97082122)

[0210] In order to facilitate blunt end cloning of the ITR sequences, wild-type Ad5 DNA was treated with Klenow enzyme in the presence of excess dNTPs. After inactivation of the Klenow enzyme and purification by phenol/chloroform extraction followed by ethanol precipitation, the DNA was digested with BamH I. This DNA preparation was used without further purification in a ligation reaction with pBr322 derived vector DNA prepared as follows: pBr322 DNA was digested with EcoR V and BamHI, dephosphorylated by treatment with TSAP enzyme (Life Technologies) and purified on LMP agarose gel (SeaPlaque GTG). After transformation into competent E. coli DH5α (Life Techn.) and analysis of ampicillinresistant colonies, one clone was selectedthat showed a digestion pattern as expected for an insert extending from the BamHI site in Ad5 to the right ITR. Sequence analysis of the cloning border at the right ITR revealed that the most 3′ G residue of the ITR was missing, the remainder of the ITR was found to be correct. The missing G residue is complemented by the other ITR during replication.

[0211] pBr/Ad.Sal-rITR (ECACC Deposit P97082119)

[0212] pBr/Ad.Bam-rITR was digested with BamH I and Sal I. The vector fragment including the adenovirus insert was isolated in LMP agarose (SeaPlaque GTG) and ligated to a 4.8 kb Sal I-BamHI fragment obtained from wt Ad5 DNA and purified with the Geneclean II kit (Bio 101, Inc.). One clone was chosen and the integrity of the Ad5 sequences was determined by restriction enzyme analysis. Clone pBr/Ad.Sal-rITR contains adeno type 5 sequences from the Sal I site at bp 16746 up to and including the rITR (missing the most 3′ G residue).

[0213] pBr/Ad.Cla-Bam (ECACC Deposit P97082117)

[0214] wt Adeno type 5 DNA was digested with Cla I and BamH I, and the 20.6 kb fragment was isolated from gel by electro-elution. pBr322 was digested with the same enzymes and purified from agarose gel by Geneclean. Both fragments were ligated and transformed into competent DH5α. The resulting clone pBr/Ad.Cla-Bam was analyzed by restriction enzyme digestion and shown to contain an insert with adenovirus sequences from bp 919 to 21566.

[0215] uBr/Ad.Afl 11-Bam (ECACC Deposit P97082114)

[0216] Clone pBr/Ad.Cla-Bam was linearized with EcoR I (in pBr322) and partially digested with Afl II. After heat inactivation of Afl II for 20 minutes at 65° C., the fragment ends were filled in with Klenow enzyme. The DNA was then ligated to a blunt double stranded oligo linker containing a Pac I site (5′-AATTGTCTTAATTAACCGCTTAA-3′) (SEQ. ID. NO.21). This linker was made by annealing the following two oligonucleotides: 5′-AATTGTCTTAATTAACCGC-3′ (SEQ. ID. NO. 22) and 5′-AATTGCGGTTAATTAAGAC-3′ (SEQ. ID. NO. 23), followed by blunting with Klenow enzyme. After precipitation of the ligated DNA to change buffer, the ligations were digested with an excess Pac I enzyme to remove concatameres of the oligo. The 22016 bp partial fragment containing Ad5 sequences from bp 3534 up to 21566 and the vector sequences, was isolated in LMP agarose (SeaPlaque GTG), religated and transformed into competent DH5α. One clone that was found to contain the Pac I site and that had retained the large adeno fragment was selected and sequenced at the 5′ end to verify correct insertion of the Pac I linker in the (lost) Afl II site.

[0217] pBr/Ad.Bam-rITRpac#2 (ECACC Deposit P97082120) and pBr/Ad.Bam-rITR#8 (ECACC Deposit P97082121)

[0218] To allow insertion of a Pac I site near the ITR of Ad5 in clone pBr/Ad.Bam-rITR about 190 nucleotides were removed between the Cla I site in the pBr322 backbone and the start of the ITR sequences. This was done as follows: pBr/Ad.Bam-rITR was digested with Cla I and treated with nuclease Bal31 for varying lengths of time (2′, 5′, 10′ and 15′). The extent of nucleotide removal was followed by separate reactions on pBr322 DNA (also digested at the Cla I site), using identical buffers and conditions. Bal31 enzyme was inactivated by incubation at 75° C. for 10 minutes, the DNA was precipitated and resuspended in a smaller volume TE buffer. To ensure blunt ends, DNAs were further treated with T4 DNA polymerase in the presence of excess dNTPs. After digestion of the (control) pBr322 DNA with Sall, satisfactory degradation (˜150 bp) was observed in the samples treated for 10 minutes or 15 minutes. The 10 minutes or 15 minutes treated pBr/Ad.Bam-rITR samples were then ligated to the above described blunted PacI linkers (see, pBr/Ad.AflII-Bam). Ligations were purified by precipitation, digested with excess Pac I and separated from the linkers on an LMP agarose gel. After religation, DNAs were transformed into competent DH5α and colonies analyzed. Ten clones were selected that showed a deletion of approximately the desired length and these were further analyzed by T-track sequencing (T7 sequencing kit, Pharmacia Biotech). Two clones were found with the Pac I linker inserted just downstream of the rITR. After digestion with Pac I, clone #2 has 28 bp and clone #8 has 27 bp attached to the ITR.

[0219] pWE/Ad.AflII-rITR (ECACC Deposit P97082116)

[0220] Cosmid vector pWE15 (Clontech) was used to clone larger Ad5 inserts. First, a linker containing a unique Pac I site was inserted in the EcoR I sites of pWE15 creating pWE.pac. To this end, the double stranded Pac I oligo as described for pBr/Ad.Afl II-BamH I was used but now with its EcoR I protruding ends. The following fragments were then isolated by electro-elution from agarose gel: pWE.pac digested with Pac I, pBr/Afl II-Bam digested with Pac I and BamHI and pBr/Ad.Bam-rITR#2 digested with BamHI and Pac I. These fragments were ligated together and packaged using λ phage packaging extracts (Stratagene) according to the manufacturer's protocol. After infection into host bacteria, colonies were grown on plates and analyzed for presence of the complete insert. pWE/Ad.Afl II-rITR contains all Ad5 sequences from bp 3534 (Afl II site) up to and including the right ITR (missing the most 3′ G residue).

[0221] pBr/Ad.IITR-Sal(9.4) (ECACC Deposit P97082115)

[0222] Adeno 5 wt DNA was treated with Klenow enzyme in the presence of excess dNTPs and subsequently digested with Sal I. Two of the resulting fragments, designated left ITR-Sal(9.4) and Sal(16.7)-right ITR, respectively, were isolated in LMP agarose (Seaplaque GTG). pBr322 DNA was digested with EcoR V and Sal I and treated with phosphatase (Life Technologies). The vector fragment was isolated using the Geneclean method (BIO 101, Inc.) and ligated to the Ad5 SalI fragments. Only the ligation with the 9.4 kb fragment gave colonies with an insert. After analysis and sequencing of the cloning border a clone was chosen that contained the full ITR sequence and extended to the Sal I site at bp 9462.

[0223] pBr/Ad.IITR-Sal(16.7) (ECACC Deposit P97082118)

[0224] pBr/Ad.IITR-Sal(9.4) is digested with Sal I and dephosphorylated (TSAP, Life Technologies). To extend this clone up to the third Sal I site in Ad5, pBr/Ad.Cla-Bam was linearized with BamH I and partially digested with Sal I. A 7.3 kb Sal I fragment containing adenovirus sequences from 9462-16746 was isolated in LMP agarose gel and ligated to the Sal I-digested pBr/Ad.IITR-Sal(9.4) vector fragment.

[0225] pWE/Ad.Afl II-EcoR I

[0226] pWe.pac was digested with Cla I and the 5′ protruding ends were filled in using Klenow enzyme. The DNA was then digested with Pac I and isolated from agarose gel. pWE/Afl II-rITR was digested with EcoR I and after treatment with Klenow enzyme digested with Pac I. The large 24 kb fragment containing the adenoviral sequences was isolated from agarose gel and ligated to the Cla I-digested and blunted pWE.pac vector using the Ligation ExpreSSTm kit from Clontech. After transformation ofUltracompetent XL 10-Gold cells from Stratagene, clones were identified that contained the expected insert. pWE/Afl II-EcoR I contains Ad5 sequences from bp 3534-27336.

[0227] B. Construction of New Adapter Plasmids

[0228] The absence of sequence overlap between the recombinant adenovirus and E1 sequences in the packaging cell line is essential for safe, RCA-free generation and propagation of new recombinant viruses. The adapter plasmid pMLPI.TK (FIG. 10) is an example of an adapter plasmid designed for use according to the invention in combination with the improved packaging cell lines of the invention. This plasmid was used as the starting material to make a new vector in which nucleic acid molecules comprising specific promoter and gene sequences can be easily exchanged.

[0229] First, a PCR fragment was generated from pZipΔMo+PyF101 (N⁻) template DNA (described in PCT/NL96/00195) with the following primers: LTR-1: 5′-CTG TAC GTA CCA GTG CAC TGG CCT AGG CAT GGA AAA ATA CAT AAC TG-3′ (SEQ. ID. NO. 24) and LTR-2: 5′-GCG GAT CCT TCG AAC CAT GGT AAG CTT GGT ACC GCT AGC GTT AAC CGG GCG ACT CAG TCA ATC G-3′ (SEQ. ID. NO.25). Pwo DNA polymerase (Boehringer Mannheim) was used according to the manufacturer's protocol with the following temperature cycles: once 5 minutes at 95° C.; 3 minutes at 55° C.; and 1 minute at 72° C., and 30 cycles of 1 minute at 95° C., 1 minute at 60° C., 1 minute at 72° C., followed by once 10 minutes at 72° C. The PCR product was then digested with BamHI and ligated into a pMLP10 (Levrero et al., Gene 101:195-202 (1991)) vector digested with Pvu II and BamH I, thereby generating vector pLTR10. This vector contains adenoviral sequences from bp 1 up to bp 454 followed by a promoter which includes part of the Mo-MuLV LTR in which the wild-type enhancer sequences are replaced by the enhancer from a mutant polyoma virus (PyF101). The promoter fragment was designated L420.

[0230] Next, the coding region of the murine HSA gene was inserted. pLTR10 was digested with BstB I followed by Klenow treatment and digestion with Nco I. The HSA gene was obtained by PCR amplification on pUC18-HSA (Kay et al, (1990) J. Immunol. 145:1952-1959) using the following primers: HSA1, 5′-GCG CCA CCA TGG GCA GAG CGA TGG TGG C-3′(SEQ. ID. NO. 26) and HSA2, 5′-GTT AGA TCT AAG CTT GTC GAC ATC GAT CTA CTA ACA GTA GAG ATG TAG AA-3′ (SEQ. ID. NO. 27). The 269 bp amplified fragment was subcloned in a shuttle vector using the Nco I and Bgl II sites. Sequencing confirmed incorporation of the correct coding sequence of the HSA gene, but with an extra TAG insertion directly following the TAG stop codon. The coding region of the HSA gene, including the TAG duplication was then excised as a Nco I(sticky)-Sal I(blunt) fragment and cloned into the 3.5 kb Nco I(sticky)/BstB I(blunt) fragment from pLTR10, resulting in pLTR-HSA 10.

[0231] Finally, pLTR-HSA10 was digested with EcoR I and BamH I after which the fragment containing the left ITR, packaging signal, L420 promoter and HSA gene was inserted into vector pMLPI.TK digested with the same enzymes, thereby replacing the promoter and the gene sequences. This resulted in the new adapter plasmid pAd/L420-HSA (FIG. 21) that contains convenient recognition sites for various restriction enzymes around the promoter and gene sequences. SnaB I and Avr II can be combined with Hpa I, Nhe I, Kpn I, Hind III to exchange promoter sequences, while the latter sites can be combined with the Cla I or BamH I sites 3′ from the HSA coding region to replace genes in this construct.

[0232] Another adapter plasmid that was designed to allow easy exchange of nucleic acid molecules was made by replacing the promoter, gene and poly A sequences in pAd/L420-HSA with the CMV promoter, a multiple cloning site, an intron and a poly-A signal. For this purpose, pAd L420-HSA was digested with Avr II and Bgl II, followed by treatment with Klenow to obtain blunt ends. The 5.1 kb fragment with pBr322 vector and adenoviral sequences was isolated and ligated to a blunt 1570 bp fragment from pcDNA1/amp (Invitrogen) obtained by digestion with Hha I and Avr II followed by treatment with T4 DNA polymerase. This adapter plasmid was named pCLIP (FIG. 22).

[0233] C. Generation of Recombinant Adenoviruses

[0234] E1-deleted Recombinant Adenoviruses with wt E3 Sequences

[0235] To generate E1 deleted recombinant adenoviruses with the new plasmid-based system, the following constructs were prepared: an adapter construct containing the expression cassette with the gene of interest linearized with a restriction enzyme that cuts at the 3′ side of the overlapping adenoviral genome fragment, preferably not containing any pBr322 vector sequences; and a complementing adenoviral genome construct pWE/Ad.AflII-rITR digested with Pac I.

[0236] These two DNA molecules are further purified by phenol/chloroform extraction and EtOH precipitation. Co-transfection of these plasmids into an adenovirus packaging cell line, preferably a cell line according to the invention, generates recombinant replication deficient adenoviruses by a one-step homologous recombination between the adapter and the complementing construct (FIG. 23). Alternatively, instead of pWE/Ad.AflII-rITR other fragments can be used, for example, pBr/Ad.Cla-Bam digested with EcoR I and BamHI or pBr/Ad.Afl III-BamHI digested with Pac I and BamHI can be combined with pBr/Ad.Sal-rITR digested with Sal I.

[0237] Recombinant adenovirus can be produced following introduction of the plasmids in the cell. It is to be understood that those skilled in the art may use other combinations of adapter and complementing plasmids without departing from the present invention.

[0238] A general protocol as outlined hereinafter, and meant as a non-limiting example of the present invention, has been performed to produce several recombinant adenoviruses using various adapter plasmids and the Ad.Afl II-rITR fragment. Adenovirus packaging cells (PER.C6) were seeded in ˜25 cm² flasks and the next day when they were at ˜80% confluence, were transfected with a mixture of DNA and lipofectamine agent (Life Techn.) as described by the manufacturer. Routinely, 40 μl lipofectamine, 4 μg adapter plasmid and 4 μg of the complementing adenovirus genome fragment AflII-rITR (or 2 μg of all three plasmids for the double homologous recombination) were used. Under these conditions transient transfection efficiencies of ˜50% (48 hrs post transfection) were obtained as determined with control transfections using a pAd/CMV-LacZ adapter. Two days later, cells were passaged to ˜80 cm² flasks and further cultured. Approximately five (for the single homologous recombination) to eleven days (for the double homologous recombination) later a cytopathic effect (CPE) was seen, indicating that functional adenovirus has formed. Cells and medium are harvested upon full CPE and recombinant virus is released by freeze-thawing. An extra amplification step in a 80 cm² flask was routinely performed to increase the yield since at the initial stage the titers was found to be variable despite the occurrence of full CPE. After amplification, viruses was harvested and plaque purified on PER. C6 cells. Individual plaques was tested for viruses with active transgenes.

[0239] Four different recombinant adenoviruses, containing the human interleukin-3 gene (see FIG. 1, PCT International Patent Application WO88/04691), the human endothelial nitric oxide gene (Janssens et al., ) J. Biol. Chem. 267:14519-14522), the TclA transposase gene (Vos et al., (1993) Genes Dev. 7:1244-1253), or the bacterial LacZ gene (Kalderon et al., (1984) Cell 39:499-509, have been produced using this protocol. In all cases, functional adenovirus was formed and all isolated plaques contained viruses with an active transgene.

[0240] E1-deleted Recombinant Adenoviruses with Modifications in the E3 or E4 Regions

[0241] Besides replacements in the E1 region, it is possible to delete the E3 region or replace part of the E3 region in the adenovirus because E3 functions are not necessary for the replication, packaging, and infection of a recombinant virus. This creates the opportunity to use a larger insert or to insert more than one gene without exceeding the maximum packagable size (approximately 105% of wt genome length). This can be done, for example, by deleting part of the E3 region in the pBr/Ad.Bam-rITR clone by digestion with Xba I and religation. This removes Ad5 wt sequences 28592-30470 including all known E3 coding regions. Another example is the precise replacement of the coding region of gp19K in the E3 region with a polylinker allowing insertion of new sequences. This leaves all other coding regions intact, obviates the need for a heterologous promoter since the transgene is driven by the E3 promoter and pA sequences, leaves more space for coding sequences and results in very high transgene expression that is at least as good as in a control E1 replacement vector.

[0242] To this end, the 2.7 kb EcoR I fragment from wt Ad5 containing the 5′ part of the E3 region was cloned into the EcoR I site of pBluescript (KS) (Stratagene). Next, the Hind III site in the polylinker was removed by digestion with EcoR V and Hinc II and subsequent religation. The resulting clone pBS.Eco-Eco/ad5ΔHIII was used to delete the gp19K coding region. Primers 1 (5′-GGG TAT TAG GCC AA AGG CGC A-3′) (SEQ. ID. NO.28) and 2 (5′-GAT CCC ATG GAA GCT TGG GTG GCG ACC CCA GCG-3′) (SEQ. ID. NO.29) were used to amplify a sequence from pBS.Eco-Eco/ad5ΔHIII corresponding to sequences 28511 to 28734 in wt Ad5 DNA. Primers 3 (5′-GAT CCC ATG GGG ATC CTT TAC TAA GTT ACA AAG CTA-3′) (SEQ. ID. NO. 30) and 4 (5′-GTC GCT GTA GTT GGA CTG G-3′) (SEQ. ID. NO. 31) were used on the same DNA to amplify Ad5 sequences from 29217 to 29476. The two resulting PCR fragments were ligated together by virtue of the newly introduced Nco I site and subsequently digested with Xba I and Mun I. This fragment was then ligated into a pBS.Eco-Eco/ad5ΔHIII vector that had been partially digested with Xba I and Mun I, generating pBS.Eco-Eco/ad5ΔHIII.Δgp19K.

[0243] To allow insertion of foreign genes into the Hind III and BamH I site, an XbaI deletion was made in pBS.Eco-Eco/ad5ΔHIII.Δgp19K to remove the BamHI sites in the Bluescript polylinker. The resulting plasmid pBS.Eco-Eco/ad5ΔHIIIΔgp19KΔXbaI, contains unique Hind III and BamHI sites corresponding to sequences 28733 (Hind III) and 29218 (BamHI) in Ad5. After introduction of a foreign gene into these sites, either the deleted XbaI fragment is re-introduced, or the insert is recloned into pBS.Eco-Eco/ad5ΔHIII.Δgg19K using Hind III and, for example Mun I. Using this procedure, we have generated plasmids expressing HSV-TK (McKnight Nucl. Acid. Res. 8:5949-5964 (1980) and Vincent et al. Hum. Gene Ther. 7:197-205 (1996)), hIL-1α (Esandi et al., (1998) Gene Therapy 5:778-788), rat IL-3β (Esandi et al., (1998) Gene 211(1):151-158), luciferase (DeWit et al., (1987) Mol. Cell Biol. 7:725-737) or LacZ. The unique Srf I and Not I sites in the pBS.Eco-Eco/ad5ΔHIII.Δgp19K plasmid (with or without an inserted gene of interest) are used to transfer the region containing the gene of interest into the corresponding region of pBr/Ad.Bam-rITR, yielding construct pBr/Ad.Bam-rITRΔgp19K (with or without an inserted gene of interest). This construct is used as described supra to produce recombinant adenoviruses. In the viral context, expression of inserted genes is driven by the adenoviral E3 promoter.

[0244] Recombinant viruses that are both E1-and E3-deleted are generated by a double homologous recombination procedure as previously described for E1-replacement vectors using a plasmid-based system which includes: an adapter plasmid for E1 replacement according to the invention, with or without insertion of a first gene of interest, the pWE/Ad.AflII-EcoR I fragment, and the pBr/Ad.Bam-rITRΔgp19K plasmid with or without insertion of a second gene of interest.

[0245] In a non-limiting example we describe the generation and functionality of a recombinant adenovirus containing the murine HSA gene in the E1 region and the firefly luciferase gene in the gp19K region. The luciferase gene was excised from pAd/MLP-Luc (described in EP 0707071) as a Hind III-BamH I construct and cloned into the Hind III-BamH I sites of pBS.Eco-Eco/ad5ΔHIIIΔgp19KΔXbaI. Then the Msc I-Mun I fragment containing the luciferse gene was cloned into the corresponding sites of pBS.Eco-Eco/ad5Δgp19K generating pBS.Eco-Eco/ad5Δgp19K.luc. This restores the Eco-Eco fragment, but now with the luciferase gene in the place of gp 19K.

[0246] To simplify further manipulation, the internal EcoR I sites in the luciferase insert were mutated without making changes to the amino acid sequence of the luciferase gene. One EcoR I site flanked the Hind III site in the 5′ non-coding region of the luciferase insert and the other one was located 588 bp 3′ from the starting ATG. A 695 bp PCR product was generated with the following primers: 5′-CGA TAA GCT TAA TTC CTT TGT OTT T-3′ (SEQ. ID. NO: 46) and 5′-CTT AGG TAA CCC AGT AGA TCC AGA GGA GTT CAT-3′ (SEQ. ID. NO: 47) and digested with Hind III and BstE II. This fragment was then ligated to Hind III-BstE II digested pBS.Eco-Eco/ad5Δgp19K.luc, replacing the corresponding insert in this vector. The resulting construct is named pBS.Eco-Eco/ad5Δgp19K.luc². The luciferase gene and part of the E3 region was then excised from this clone with Srf I and Not I and introduced in the corresponding sites in pBr/Ad.Bam-rITR generating clone pBr/Ad.Bam-rITRΔgp19K/luc².

[0247] The adapter plasmid pAd5/Si800HSA used for the replacement of E1 in the double insert virus contains the murine HSA gene driven by a retrovirus LTR-based promoter. This adapter plasmid was generated from the pAd5/L420-HSA construct described infra by replacement of the promoter sequence. First a PCR product was generated on a retroviral vector based on the MFG-S vector described in WO 95/34669 using the same primers as for the amplification of the L420 promoter fragment (described infra). This PCR amplifies the sequences corresponding to bp 453-877 in the MFG-S vector. The L420 promoter in pAd5/L420-HSA (FIG. 21) was then exchanged for the PCR fragment using the unique Avr II and Hind II sites. The resulting construct, pAd5/S430-HSA, was then digested with Nhe I and Sca I and the 4504 bp fragment containing the HSA gene, pA sequences, Ad5 sequences and vector sequences to the Sca I site in the ampicillin gene was isolated.

[0248] The construct pAd5/S430-HSA also was digested with Xba I and Sca I and the 1252 bp fragment (containing the remainder of the ampicillin gene, the left ITR and packaging signal from adenovirus and the 5′ part of the S430 promoter) was isolated. A third fragment of 1576 bp was isolated from the MFG-S-based retroviral vector following an XbaI digestion and contains MFG-S sequences corresponding to bp 695-2271.

[0249] The adapter plasmid pAd5/S1800-HSA was constructed by ligating the three isolated fragments. The double insert virus Ad5/S1800-HSA.E31uc was generated (as described above) by transfection of the following DNA fragments into PER.C6 cells: pAd5/Si800-HSA digested with EcoR I and Sal 1 (2 μg). At occurrence of CPE, the virus was harvested and amplified by serial passages on PER.C6 cells. The activity of this HSA-Luc virus was compared to single insert ΔE1 viruses containing either the S1800-HSA or the CMV-Luc transcription units in the E1 region. A549 cells were seeded at 2×10⁵ cells/well and infected 5 hrs later with different amounts of the virus. Two days later transgene expression was measured. Luciferase activity was measured using a luciferase assay system (Promega) and expression of the murine HSA gene was measured with an α-HSA antibody (M1/69, Pharmingen). The results are listed in Table III.

[0250] This experiment shows that using the plasmid-based recombination system, double insert viruses can be made and that both inserts are functional. Furthermore, the luciferase activity of the double insert viruses is comparable to the CMV-driven luciferase activity of the control virus. Therefore, we conclude that the E3 promoter is highly active in A549 cells, even in the absence of E1A proteins.

[0251] In addition to manipulations in the E3 region, changes of (parts of) the E4 region can be accomplished easily in pBr/Ad.Bam-rITR. Generation and propagation of such a virus, however, in some cases demands complementation in trans.

Example 3

[0252] Demonstration of the Competence of a Synthetic DNA Sequence, That is Capable of Forming a Hairpin Structure, to Serve as a Primer for Reverse Strand Synthesis for the Generation of Double-stranded DNA Molecules in Cells that Contain and Express Adenoviral Genes.

[0253] Name convention of the plasmids used:

[0254] p plasmid

[0255] I ITR (Adenoviral Inverted Terminal Repeat)

[0256] C Cytomegalovirus (CMV) Enhancer/Promoter Combination

[0257] L Firefly Luciferase Coding Sequence

[0258] hac, haw Potential hairpin that can be formed after digestion with restriction endonuclease Asp718 in both the correct and in the reverse orientation, respectively (FIG. 15)

[0259]FIG. 15 depicts a potential hairpin conformation of a single-stranded DNA molecule(5′-GTACACTGACCTAGTGCCGCCCGGGCAAAGCCCGGGCGGCACTAG-3′(SEQ. ID. NO.45) that contains the HP/asp sequence. Restriction with the endonuclease Asp718 of plasmid pICLhaw, containing the annealed oligonucleotide pair HP/asp1 and HP/asp2 will yield a linear double-stranded DNA fragment. In cells in which the required adenovirus genes are present, replication can initiate at the terminus that contains the ITR sequence. During the chain elongation, one of the strands will be displaced. The terminus of the single-stranded displaced strand molecule can adopt the conformation depicted in FIG. 15. In this conformation, the free 3′-terminus can serve as a primer for the cellular and/or adenoviral DNA polymerase, resulting in conversion of the displaced strand in a double-stranded form.

[0260] The naming convention is exemplified as follows. pICLhaw is a plasmid that contains the adenovirus ITR followed by the CMV-driven luciferase gene and the Asp718 hairpin in the reverse (non-functional) orientation.

[0261] Plasmids pICLhac, pICLhaw, pICLI and pICL were generated using standard techniques. The schematic representation of these plasmids is shown in FIGS. 16-19.

[0262] Plasmid pICL is derived from the following plasmids: nt.1 457 pMLP10 (Levrero et al., (1991) Gene 101:195-202) nt.458 1218 pCMVβ (Clontech, EMBL Bank No. U02451) nt.1219 3016 pMLP.luc (IntroGene, unpublished) nt.3017 5620 pBLCAT5 (Stein et al., (1989) Mol. Cell Biol. 9:4531-4).

[0263] The plasmid has been constructed as follows:

[0264] The tet gene of plasmid pMLP10 has been inactivated by deletion of the BamH I-Sal I fragment, to generate pBLP10ΔSB. Using primer set PCR/MLP1 and PCR/MLP3 a 210 bp fragment containing the Ad5-ITR, flanked by a synthetic Sal I restriction site was amplified using pMLP10 DNA as the template. The PCR product was digested with the enzymes EcoR I and SgrA I to generate a 196 bp fragment. Plasmid pMLP10ΔSB was digested with EcoR I and SgrA I to remove the ITR. This fragment was replaced by the EcoR I-SgrA I-treated PCR fragment to generate pMLP/SAL.

[0265] Plasmid pCMV-Luc was digested with Pvu II to completion and recirculated to remove the SV40-derived poly-adenylation signal and Ad5 sequences with exception of the Ad5 left-terminus. In the resulting plasmid, pCMV-lucΔAd, the Ad5 ITR was replaced by the Sal-site-flanked ITR from plasmid pMLP/SAL by exchanging the Xmn I-Sac II fragments. The resulting plasmid, pCMV-lucΔAd/SAL, the Ad5 left terminus and the CMV-driven luciferase gene were isolated as a Sal I-Sma I fragment and inserted in the Sal I and Hpa I digested plasmid pBLCATS, to form plasmid pICL. Plasmid pICL is represented in FIG. 19; its sequence is presented in FIG. 20.

[0266] Plasmid pICL contains the following features: nt.1-457 Ad5 left terminus (Sequence 1-457 of Ad 5) nt.458-969 Human cytomegalovirus enhancer and immediate early promoter (Boshart et al., (1985) Cell 41:521-530) (from plasmid pCMVβ, Clontech, Palo Alto, USA) nt.970-1204 SV40 19S exon and truncated 16/19S intron (from plasmid pCMVβ) nt.1218-2987 Firefly luciferase gene (from pMLP.luc) nt.3018-3131 SV40 tandem poly-adenylation signals from late transcript, derived from plasmid pBLCAT5) nt.3132-5620 pUC12 backbone (derived from plasmid pBLCAT5) nt.4337-5191 β-lactamase gene (Amp-resistance gene, reverse orientation)

[0267] Plasmids pICLhac and pICLhaw

[0268] Plasmids pICLhac and pICLhaw were derived from plasmid pICL by digestion of pICL with the restriction enzyme Asp718. The linearized plasmid was treated with Calf-Intestine Alkaline Phosphatase to remove the 51 phosphate groups. The partially complementary synthetic single-stranded oligonucleotides Hp/asp1 and Hp/asp2 were annealed and phosphorylated on their 5′ ends using T4-polynucleotide kinase.

[0269] The phosphorylated double-stranded oligomers were mixed with the dephosphorylated pICL fragment and ligated. Clones containing a single copy of the synthetic oligonucleotide inserted into the plasmid were isolated and characterized using restriction enzyme digests. Insertion of the oligonucleotide into the Asp718 site will at one junction recreate an Asp718 recognition site, whereas at the other junction the recognition site will be disrupted. The orientation and the integrity of the inserted oligonucleotide was verified in selected clones by sequence analyses. A clone containing the oligonucleotide in the correct orientation (the Asp718 site close to the 3205 EcoR I site) was denoted pICLhac. A clone with the oligonucleotide in the reverse orientation (the Asp718 site close to the SV40 derived poly signal) was designated pICLhaw. Plasmids pICLhac and pICLhaw are represented in FIGS. 16 and 17.

[0270] Plasmid pICLI was created from plasmid pICL by insertion of the Sal I-SgrA I fragment from pICL, containing the Ad5-ITR into the Asp718 site of pICL. The 194 bp Sal I-SgrA I fragment was isolated from pICL, and the cohesive ends were converted to blunt ends using E. coli DNA polymerase I (Klenow fragment) and dNTP's. The Asp718 cohesive ends were converted to blunt ends by treatment with mungbean nuclease. By ligation clones were generated that contain the ITR in the Asp718 site of plasmid pICL. A clone that contained the ITR fragment in the correct orientation was designated pICLI (FIG. 18).

[0271] Generation of Adenovirus Ad-CMV-hcTK.

[0272] Recombinant adenovirus was constructed according to the method described in EPO Patent Application 95202213. Two components are required to generate a recombinant adenovirus. First, an adaptor-plasmid containing the left terminus of the adenovirus genome containing the ITR and the packaging signal, an expression cassette with the gene of interest, and a portion of the adenovirus genome which can be used for homologous recombination. In addition, adenovirus DNA is needed for recombination with the aforementioned adaptor plasmid. In the case of Ad-CMV-hcTK, the plasmid PCMV.TK was used as a basis. This plasmid contains nt.1-455 of the adenovirus type 5 genome, nt. 456-1204 derived from pCMVβ (Clontech, the Pst I-Stu I fragment that contains the CMV enhancer promoter and the 16S/19S intron from simian Virus 40), the HSV-TK gene (described in EPO Patent Application 95202213.5), the SV40-derived polyadenylation signal (nt.2533-2668 of the SV40 sequence), followed by the Bgl II-Sca I fragment of Ad5 (nt. 3328-6092 of the Ad5 sequence). These fragments are present in a pMLP10-derived (Levrero et al., (1991) Gene 101: 195-202) backbone. To generate plasmid pAD-CMVhc-TK, plasmid pCMV.TK was digested with Cla I (the unique Cla I-site is located just upstream of the TK open reading frame) and dephosphorylated with Calf-Intestine Alkaline Phosphate.

[0273] To generate a hairpin-structure, the synthetic oligonucleotides HP/cla2 and HP/cla2 were annealed and phosphorylated on their 5′ -OH groups with T4-polynucleotide kinase and ATP. The double-stranded oligonucleotide was ligated with the linearized vector fragment and used to transform E. coli strain “Sure”. Insertion of the oligonucleotide into the Cla I site will disrupt the Cla I recognition sites. The oligonucleotide contains a new Cla I site near one of its termini. In selected clones, the orientation and the integrity of the inserted oligonucleotide was verified by sequence analyses. A clone containing the oligonucleotide in the correct orientation (the Cla I site at the ITR side) was denoted pAd-CMV-hcTK. This plasmid was co-transfected with Cla I-digested wild-type adenovirus-type5 DNA into 911 cells. A recombinant adenovirus in which the CMV-hcTK expression cassette replaces the E1 sequences was isolated and propagated using standard procedures.

[0274] To study whether the hairpin can be used as a primer for reverse strand synthesis on the displaced strand after replication has started at the ITR, the plasmid pICLhac was introduced into 911 cells, i.e. HER transformed with the adenovirus E1 region. The plasmid pICLhaw served as a control: it contains the oligonucleotide pair HP/asp 1 and 2 in the reverse orientation but is otherwise completely identical to plasmid pICLhac. Also included in these studies were plasmids pICLI and pICL. In the plasmid pICLI, the hairpin is replaced by an adenovirus ITR. Plasmid pICL contains neither a hairpin nor an ITR sequence. These plasmids served as controls to determine the efficiency of replication by virtue of the terminal hairpin structure. To provide the viral products other than the E1 proteins (these are produced by the 911 cells) required for DNA replication, the cultures were infected with the virus IG.Ad.MLPI.TK after transfection. Several parameters were being studied to demonstrate proper replication of the transfected DNA molecules. First, DNA extracted from the cell cultures transfected with the aforementioned plasmids and infected with IG.Ad.MLPI.TK virus was analyzed by Southern blotting for the presence of the expected replication intermediates, as well as for the presence of the duplicated genomes. Furthermore, from the transfected and IG.Ad.MLPI.TK infected cell populations, virus was isolated that can transfer a luciferase marker gene into luciferase negative cells and express it.

[0275] Plasmid DNA of plasmids pICLhac, pCLhaw, pICLI and pICL were digested with restriction endonuclease Sal I and treated with mungbean nuclease to remove the 4 nucleotide single-stranded extension of the resulting DNA fragment. In this manner, a natural adenovirus 5′ ITR terminus on the DNA fragment was created. Subsequently, both the pICLhac and pICLhaw plasmids were digested with restriction endonuclease Asp718 to generate the terminus capable of forming a hairpin structure. The digested plasmids were introduced into 911 cells, using the standard calcium phosphate co-precipitation technique, four dishes for each plasmid. During the transfection, for each plasmid two of the cultures were infected with the IG.Ad.MLPI.TK virus using 5 infectious IG.Ad.MLPI.TK particles per cell. At twenty-hours post transfection and forty hours post-transfection one Ad.tk-virus-infected and one uninfected culture were used to isolate low molecular-weight DNA using the procedure devised by Hirt (as described in Einerhand et al., (1995) Gene Therapy 2:336-343). Aliquots of isolated DNA were used for Southern analysis. After digestion of the samples with restriction endonuclease EcoR I using the luciferase gene as a probe a hybridizing fragment of approx. 2.6 kb were detected in only the samples from the adenovirus-infected cells transfected with plasmid pICLhac. The size of this fragment was consistent with the anticipated duplication of the luciferase marker gene. This supports the conclusion that the inserted hairpin is capable of serving as a primer for reverse strand synthesis. The hybridizing fragment was absent if the IG.Ad.MLPI.TK virus was omitted, or if the hairpin oligonucleotide was inserted in the reverse orientation.

[0276] The restriction endonuclease Dpn I recognizes the tetranucleotide sequence 5′-GATC-3′, but cleaves only methylated DNA, (that is, only plasmid DNA propagated in, and derived, from E. coli not DNA that has been replicated in mammalian cells). The restriction endonuclease Mbo I recognizes the same sequences, but cleaves only unmethylated DNA (namely, DNA propagated in mammalian cells). DNA samples isolated from the transfected cells are incubated with Mbo I and Dpn I and analyzed with Southern blots. These results demonstrated that only in the cells transfected with the pICLhac and the pICLI plasmids large Dpn I-resistant fragments were present, that were absent in the Mbo I treated samples. The data demonstrates that only after transfection of plasmids pICLI and pICLhac replication and duplication of the fragments occur. The data demonstrates that in adenovirus-infected cells linear DNA fragments that have on one terminus an adenovirus-derived ITR and at the other terminus, a nucleotide sequence that can anneal to sequences on the same strand, when present in single-stranded form thereby generate a hairpin structure, and will be converted to structures that have inverted terminal repeat sequences on both ends. The resulting DNA molecules will replicate by the same mechanism as the wild-type adenovirus genomes.

Example 4

[0277] Demonstration That the DNA Molecules that Contain a Luciferase Marker Gene, a Single Copy of the ITR, the Encapsulation Signal and a Synthetic DNA Sequence, That is Capable of Forming a Hairpin Structure, are Sufficient to Generate DNA Molecules That can be Encapsulated into Virions.

[0278] To demonstrate that the DNA molecules, generated in Example 3, containing two copies of the CMV-Luc marker gene can be encapsulated into virions, virus was harvested from the remaining two cultures via three cycles of freeze-thaw crushing and was used to infect murine fibroblasts. Forty-eight hours after infection, the infected cells are assayed for luciferase activity. To exclude the possibility that the luciferase activity has been induced by transfer of free DNA, rather than via virus particles, virus stocks were treated with DNaseI to remove DNA contaminants. Furthermore, as an additional control, aliquots of the virus stocks were incubated for 60 minutes at 56° C. The heat treatment does not affect the contaminating DNA, but does inactivate the viruses. Significant luciferase activity was only found in the cells after infection with the virus stocks derived from IG.Ad.MLPI.TK-infected cells transfected with the pICLhc and pICLI plasmids. Neither in the non-infected cells, nor in the infected cells transfected with the pICLhw and pICL was significant luciferase activity demonstrated. Heat inactivation, but not DNaseI treatment, completely eliminated luciferase expression, demonstrating that adenovirus particles, and not free (contaminating) DNA fragments were responsible for transfer of the luciferase reporter gene.

[0279] These results demonstrate that these small viral genomes can be encapsulated into adenovirus particles and suggest that the ITR and the encapsulation signal are sufficient for encapsulation of linear DNA fragments into adenovirus particles. These adenovirus particles can be used for efficient gene transfer. When introduced into cells that contain and express at least some of the adenovirus genes (namely E1, E2, E4, and L, and VA), recombinant DNA molecules that include at least one ITR, at least part of the encapsulation signal as well as a synthetic DNA sequence, that is capable of forming a hairpin structure, have the intrinsic capacity to autonomously generate recombinant genomes which can be encapsulated into virions. Such genomes and vector system can be used for gene transfer.

Example 5

[0280] Demonstration That DNA Molecules Which Contain Nucleotides 3510-35953 (Namely 9.7-100 Map Units) of the Adenovirus Type 5 Genome (Thus Lack the E1 Protein-coding Regions, the Right-hand ITR and the Encapsulation Sequences) and a Terminal DNA Sequence That is Complementary to a Portion of the Same Strand of the DNA Molecule When Present in Single-stranded Form Other Than the ITR, and as a Result is Capable of Forming a Hairpin Structure, can Replicate in 911 Cells.

[0281] In order to develop a replicating DNA molecule that can provide the adenovirus products required to allow the above-mentioned ICLhac vector genome and alike minimal adenovectors to be encapsulated into adenovirus particles by helper cells, the Ad-CMV-hcTK adenoviral vector was developed. Between the CMV enhancer/promoter region and the thymidine kinase gene, the annealed oligonucleotide pair (Table I) HP/cla 1 and 2 was inserted. The vector Ad-CMV-hcTK was propagated and produced in 911 cell using standard procedures. This vector was grown and propagated exclusively as a source of DNA used for transfection. DNA of the adenovirus Ad-CMV-hcTK was isolated from virus particles that had been purified using CsC 1 density-gradient centrifugation by standard techniques. The virus DNA was digested with restriction endonuclease Cla I. The digested DNA was size-fractionated on an 0.7% agarose gel and the large fragment was isolated and used for further experiments. Cultures of 911 cells were transfected with the large Cla I-fragment of the Ad-CMV-hcTK DNA using standard calcium phosphate co-precipitation techniques. Much like in the previous experiments with plasmid pICLhac, the Ad-CMV-hc replicates starting at the right-hand ITR. Once the 1-strand is displaced, a hairpin can be formed at the left-hand terminus of the fragment. This facilitates DNA polymerase elongation of the chain towards the right-hand side. The process proceeds until the displaced strand is completely converted to its double-stranded form. Finally, the right-hand ITR is recreated, and in this location, normal adenovirus replication-initiation and elongation occur. The polymerase reads through the hairpin, thereby duplicating the molecule. The input DNA molecule of 33250 bp, that had on one side an adenoviral ITR sequence and at the other side a DNA sequence that had the capacity to form a hairpin structure is duplicated so that both ends contain an ITR sequence. The resulting DNA molecule consists of a type of palindrome structure of approximately 66500 bp.

[0282] This structure is detected in low-molecular weight DNA extracted from transfected cells using Southern analysis. The palindromic nature of the DNA fragment can be demonstrated by digestion of the low-molecular weight DNA with suitable restriction endonucleases and Southern blotting with the HSV-TK gene as the probe. This molecule can replicate itself in the transfected cells by virtue of the adenoviral gene products that are present in the cells. In part, the adenoviral genes are expressed from templates that are integrated in the genome of the target cells (namely, the E1 gene products), the other genes reside in the replicating DNA fragment itself. This linear DNA fragment cannot be encapsulated into virions. Not only does it lack all the DNA sequences required for encapsulation, but its size also is much too large to be encapsulated

Example 6

[0283] Demonstration That DNA Molecules that Contain Nucleotides 3503-35953 (Viz. 9.7-100 Map Units) of the Adenovirus Type 5 Genome (Thus Lacking the e1 Protein-coding Regions, the Right-hand ITR and the Encapsulation Sequences) and a Terminal DNA Sequence That is Complementary to a Portion the Same Strand of the DNA Molecule Other Than the ITR, and as a Result is Capable of Forming a Hairpin Structure, can Replicate in 911 Cells and Can Provide the Helper Functions Required to Encapsulate the pICLI and pICLhac Derived DNA Fragments.

[0284] The purpose of the next series of experiments is to demonstrate that the DNA molecule described in Example 5 can be used to encapsulate the minimal adenovectors described in Examples 3 and 4.

[0285] The large fragment isolated after endonuclease Cla I-digestion of Ad-CMV-hcTK DNA was introduced into 911 cells (as described in Example 5) together with endonuclease Sal I mungbean nuclease, endonuclease Asp718-treated plasmid pICLhac, or as a control similarly treated plasmid pICLhaw. After 48 hours, virus was isolated by freeze-thaw crushing of the transfected cell population. The virus preparation was treated with DNaseI to remove contaminating free DNA. The virus was used subsequently to infect Rat2 fibroblasts. Forty-eight hours post infection, the cells were assayed for luciferase activity. Only in the cells infected with virus isolated from the cells transfected with the pICLhac plasmid, and not with the pICLhaw plasmid, was significant luciferase activity demonstrated. Heat inactivation of the virus prior to infection completely abolished the luciferase activity, indicating that the luciferase gene was transferred by a viral particle. Infection of 911 cell with the virus stock did not result in any cytopathological effects (CPE), demonstrating that pICLhac was produced without any infectious helper virus being propagated on 911 cells. These results demonstrate that the proposed method can be used to produce stocks of minimal-adenoviral vectors, that are completely devoid of infectious helper viruses that are able to replicate autonomously on adenovirus-transformed human cells or on non-adenovirus transformed human cells.

Example 7

[0286] Construction of Plasmids for the Generation and Production of Minimal Adenoviral Vectors.

[0287] A minimal adenoviral vector contains (as operably linked components) the adenovirus-derived cis elements necessary for replication and packaging, with or without foreign nucleic acid molecules to be transferred. Recently, the lower limit for efficient packaging of adenoviral vectors has been determined at 75% of the genome length (Parks and Graham, J. Virol. 71(4):3293-8 (1997)). To allow flexible incorporation of various lengths of stuffer fragments, a multiple cloning site (MCS) was introduced into a minimal adenoviral vector. To obtain a minimal adenoviral vector according to the invention, the following constructs were made: pAd/L420-HSA (FIG. 21) was digested with Bgl II and Sal I and the vector-containing fragment was isolated. This fragment contains the left ITR and packaging signal from Ad5 and the murine HSA gene driven by a modified retroviral LTR. The right ITR of adenovirus was amplified by PCR on pBr/Ad.BamH I-rITR template DNA using the following primers: PolyL-ITR: 5′-AAC-TGC-AGA-TCT-ATC-GAT-ACT-AGT-CAA-TTG-CTC-GAG-TCT-AGA-CTA-CGT-CAC-CCG-CCC-CGT-TCC-3′ (SEQ. ID. NO. 32) and ITR-BSN: 5′-CGG-GAT-CCG-TCG-ACG-CGG-CCG-CAT-CAT-CAA-TAA-TAT-ACC-3′ (SEQ. ID. NO. 33). The amplified fragment was digested with Pst I and BamHI and cloned into pUC 119 digested with the same enzymes. After sequence confirmation of correct amplification of the ITR and the MCS, a Bgl II-Sal I fragment was isolated and cloned into the Bgl II-Sal-digested pAd/L420-HSA fragment described above. The resulting clone was named pAd/L420-HSA.ITR.

[0288] To be able to manipulate constructs of lengths exceeding 30 kb, the minimal adenoviral vector pAd L420-HSA.ITR was subcloned in a cosmid vector background. To this end, the cosmid vector pWE15 was modified to remove restriction sites in the backbone. pWE15 was digested with Pst I and fragments of 4 kb and 2,36 kb were isolated from agarose gel and ligated together. The resulting clone, stripped of the SV40 ori/early promoter and neomycine resistance coding sequence, was named pWE20. Then, pWE20 was digested with Cla I and Hind III and the sticky ends were filled in with Klenow enzyme. A6354 bp blunt fragment was ligated to a phosphorylated Nsi I linker with the following sequence: 5′-CGATGCATCG-3′ (SEQ. ID. NO.34). The ligated DNA was phenol/chloroform extracted, precipitated with EtOH to change buffers, and digested with excess Nsi I. Digested DNA was separated from the linkers by electrophoresis, isolated and religated. The resulting clone was named pWE25. Correct insertion of the Nsi I linker was confirmed by restriction enzyme digestion and sequencing. To construct the minimal adenoviral vector, pAd/L420-HSA.ITR was digested with Sca I and Not I and the 2 kb fragment containing part of the ampicillin gene and the adeno ITRs was cloned into pWE25 digested with Sca I and Not I. The resulting clone was named pMV/L420H (FIG. 24). This clone allows easy manipulation to exchange the promoter and/or gene, and also allows insertion of DNA fragments of lengths not easily cloned into normal plasmid backbones.

[0289] Plasmid pMV/CMV-LacZ was made by exchanging the L420-HSA fragment (SnaB I-BamHI) for a fragment from pcDNA3-nlsLacZ (Nru I-BamHI) containing the CMV promoter and LacZ coding sequences. pcDNA3-nlsLacZ was constructed by insertion of a Kpn I-BamH I fragment obtained after PCR amplification of the nlsLacZ coding sequences into pcDNA3 (Invitrogen) digested with Kpn I and BamHI. The PCR reaction was performed on a pMLP.nlsLacZ template DNA using the primers 1:5′-GGG-GTG-GCC-AGG-GTA-CCT-CTA-GGC-TTT-TGC-AA-3′ (SEQ. ID. NO. 35) and 2: 5′-GGG-GGG-ATC-CAT-AAA-CAA-GTT-CAG-AAT-CC-3′ (SEQ. ID. NO.36). Correct amplification and cloning were confirmed by assaying ′3-galactosidase expression in a transient transfection experiment on 911 cells.

[0290] The vector pAd/MLPnlsLacZ was made as follows: pMLP10 (Levrero et al., (1991) Gene 101:195-202) was digested with Hind III and BamHI and ligated, in a three-part ligation, to a 3.3 kb Avr I-BamH I fragment from L7RHβgal (Kalderon et al., ) Cell 499-509), and a synthetic linker with Hind III and XbaI overhang. The linker was made by annealing two oligonucleotides of sequence 5′-AGC TTG AAT TCC CGG GTA CCT-3′ (SEQ. ID. NO.37) and 5′-CTA GAG GTA CCC GGG AAT TCA-3′ (SEQ. ID. NO. 38). The resulting clone was named pMLP.nlsLacZ/-Ad. Next, pMLP.nlsLacZ/-Ad was digested with BamHI and Nru I and the vector containing fragment was ligated to a 2766 bp Bgl I-Sca I fragment from pAd5SalB (Bemards et al., (1982) Virology 120:422-432). This resulted in the adapter plasmid pMLP.nlsLacZ (described in European Patent Office Application EP 0 707 071).

[0291] Propagation of a minimal adenoviral vector can only be achieved by expression of adenovirus gene products. Expression of adenoviral gene products, at levels high enough to sustain production of large quantities of virus, requires replication of the coding nucleic acid molecule. Therefore, replicating helper viruses are usually used to complement the minimal adenoviral vectors. The present invention, however, provides packaging systems for minimal adenoviral vectors without the use of helper viruses. One of the methods of the invention makes use of a replicating DNA molecule that contains the 5′-ITR and all adenoviral sequences betweenbp 3510 and35938, i.e., the complete adenoviral genome except for the E1 region and the packaging signal. Construct pWE/Ad.A5′ (FIG. 25) is an example of a replicating molecule according to the invention that contains two adenoviral ITRs. pWE/Ad.Δ5′. It has been made in a cosmid vector background from three fragments.

[0292] First, the 5′ ITR from Ad5 was amplified using the following primers: ITR-EPH: 5′-CGG-AAT-TCT-TAA-TTA-AGT-TAA-CAT-CAT-CAA-TAA-TAT-ACC-3′ (SEQ. ID. NO.39) and ITR-pIX: 5′-ACG-GCG-CGC-CTT-AAG-CCA-CGC-CCA-CAC-ATT-TCA-GTA-CGT-ACT-AGT-CTA-CGT-CAC-CCG-CCC-CGT-TCC-3′ (SEQ. ID. NO. 40). The resulting PCR fragment was digested with EcoR I and Asc I and cloned into vector pNEB 193 (New England Biolabs) digested with the same enzymes. The resulting construct was named pNEB/ITR-pIX. Sequencing confirmed correct amplification of the Ad5 sequences in the left ITR (Ad5 sequences 1 to 103) linked to the pIX promoter (Ad5 sequences 3511 to 3538) except for a single mismatch with the expected sequence according to GenBank (Accession no.: M73260/M29978), i.e., an extra C-residue was foundjust upstream of the Afl II site. This ITR-pIX fragment was isolated with EcoR I and Afl II and ligated to a EcoR I-Afl II vector fragment containing Ad5 sequences 3539-21567. The latter fragment was obtained by digestion of pBr/Ad.Cla-Bam (supra) with EcoR I and partially with Afl II. The resulting clone was named pAd/LITR(Δ5′)-BamH I. The final construct pWE/Ad.Δ5′ was made by ligating cosmid vector pWE15.Pac (supra) digested with Pac I to pAd/LITR(A5′)-BamH I digested with Pac I/BamH I and pBr/Ad.Bam-rITR.pac#2 (supra) digested with Pac I/BamH I (FIG. 25).

[0293] An alternative method to produce packaging systems for minimal adenoviral vectors without the use of helper viruses according to the invention is to use a replicating DNA molecule that contains the complete adenoviral genome except for the E1 region and the packaging signal and in which one of the ITRs is replaced by a fragment containing a DNA sequence complementary to a portion of the same strand other than the ITR and that therefore is able to form a hairpin structure (FIG. 15). In a non-limiting example, the DNA sequence complementary to a portion of the same strand other than the ITR is derived from the adeno-associated virus (AAV) terminal repeat. Such a replicating DNA molecule is made following the same cloning strategy as described for pWE/Ad.Δ5′, but now starting with the AAV terminal repeat linked to part of the adenoviral pIX promoter. To this end, the adenoviral ITR sequences between the Hpa I and Spe I sites in construct pNEB/ITR-pIX were exchanged for the AAV ITR by introducing the Pvu II/Xba I fragment from psub201(+) containing the AAV ITR (Samulski et al., (1989) J. Virol. 63:3822-3828). This results in construct pWE/AAV.Δ5′ that replicates in an E1 complementing cell line.

[0294] Another alternative packaging system for minimal adenoviral vectors is described infra and makes use of the replication system of SV40. A functional helper molecule according to this method contains at least the adenoviral sequences necessary to sustain packaging of a minimal construct but not the E1 sequences and packaging signal, and preferably also lacking ITRs. This adenovirus-derived entity has to be present on a vector that contains, besides the sequences needed for propagation in bacteria, an origin of replication from SV40 virus. Transfection of such a molecule together with the minimal adenoviral vector, described supra, into a packaging cell line (e.g., PER.C6) expressing, besides the E1 proteins, SV40 derived Large T antigen proteins, results in Large T-dependent replication of the adenovirus-derived helper construct. This replication leads to high levels of adenoviral proteins necessary for replication of the minimal adenoviral vector and packaging into virus particles. In this way, there is no sequence overlap that leads to homologous recombination between the minimal adenoviral vector construct and the helper molecule. In addition, there is no sequence overlap that leads to homologous recombination between the helper molecule and minimal adenoviral vector on the one side and the E1 sequence in the packaging cell on the other side.

[0295] Replication of a 40 kb adenoviral construct was investigated in cells expressing SV40 Large T proteins. Hereto, 2×10⁶Cos-1 cells were transfected in a T25 flask with the following constructs complexed with lipofectamine reagent (Life techn.): the 8 kb cosmid vector pWE.pac, the 40.5 kb construct pWE/Ad.AflII-rITR and three clones (#1, #5 and #9) of the 40.6 kb construct pWE/Ad.Δ5′ (described infra). Control transfections were carried out with the constructs pWE.pac and pWE/Ad.AflII-rITR digested with Pac I enzyme and a CMV-LacZ expression vector without the SV40 ori sequence. Transfection efficiency was 50% as determined by a separate transfection using the CMV-LacZ vector and X-gal staining after 48 hrs. All cells were harvested 48 hrs. following transfection and DNA was extracted according to the Hirt procedure (as described in Einerhand et al., (1995) Gene Therapy 2:336-343). Final pellets were resuspended in 50μl TE+RNase (20 μg/ml) and 10 μl samples were digested with Mbo 1 (35 units overnight at 37° C.). Undigested samples (5μl) and Mbo I digested samples were run on a 0.8% agarose gel, transferred to a nylon filter (Amersham) and hybridized to radioactive probes according to standard procedures. One probe was derived from an 887 bp Dpn I fragment from the cosmid vector pWE.pac and one was derived from a 1864 bp BsrG I-BamH I fragment from adenoviral sequences. These probes hybridize to a 887 bp band and a 1416 bp respectively in Mbo I digested material. Input DNA from bacterial origin is methylated and therefore not digested with Mbo I. In this way, it is possible to specifically detect DNA that is replicated in eukaryotic cells. FIG. 26 part A shows a schematic presentation of the construct pWE/Ad.Δ5′ and the locations of the SV40 origin of replication, the pWE-derived probe and the adenovirus-derived probe. The lower part presents the autoradiograms of the Southern blots hybridized to the adenovirus probe (FIG. 26, part B) and the pWE probe (FIG. 26, part C). (See legends for explanation of sample loading.) These experiments show that all lanes that contain material from Cos-1 cells that are transfected with plasmids harboring an SV40 ori contain Mbo I sensitive DNA and show a specific band of the expected length. The bands specific for replication in the lanes with Cos-1 cells transfected with Pac I digested material (lanes B 17/18 and C 15-18) probably result from incomplete Pac I digestion. From these experiments it can be concluded that it is possible to replicate large DNA fragments with the SV40 LargeT/ori system in eukaryotic cells.

Example 8

[0296] A functional adenovirus helper molecule lacking ITR sequences was constructed starting with the clone pWE/Ad.D5′ described supra. pWE/Ad.D5′ was digested with Bstl 1071 and the 17.5 kb vector-containing fragment was religated to give pWE/Ad.D5′-Bstl 1071. This clone was then used to amplify the 3′ part of the adenovirus genome sequences without the right ITR. A 2645 bp PCR fragment was generated using the primers Ad3′/Forw: 5′-CGG AAT TCA TCA GGA TAG GGC GGT GG-3′ (SEQ. ID. NO.41) and Ad3′/Rev: 5′-CGG GAT CCT ATC GAT ATT TAA ATG TTT TAG GGC GGA GTA ACT TG-3′ (SEQ. ID. NO. 42). The amplified fragment was digested with EcoR I and BamH I and subcloned in pBr322 digested with the same enzymes. After confirmation of correct amplification by sequencing, the 2558 bp Sbf I-Cla I fragment of this clone was recloned in pWE/Ad.D5′-Bstl 1071 digested with the same enzymes. The resulting construct lacks the right ITR and is named pWE/ΔrI-Bstl 1071. Next, in this clone the left ITR was replaced by a linker with a Pac I and Afl II overhang made up by annealing the following primers: PA-pIXI 5′-TAA GCC ACT AGT ACG TAC TGA AAT GTG TGG GCG TGG C-3′ (SEQ. ID. NO. 43) and PA-pIX 5′-TTA AGC CAC GCC CAC ACA TTT CAG TAC GTA CTA GTG GCT TAA T-3′ (SEQ. ID. NO. 44). This removed the left ITR and restored correct sequence of the pIX promoter. The clone is named pWE/ΔITR-Bstl 1071. Correct insertion of the double stranded linker was confirmed by sequencing. The deleted Bstl 1071 fragment was then cloned back into pWE/AITR-Bstl 1071 and the correct orientation was checked by restriction digestion. The resulting clone is named pWE/Ad-H. Following transfection of this DNA molecule into packaging cells that express adenoviral E1 proteins and the SV40 Large T antigen, replication of that molecule takes place resulting in high levels of adenoviral proteins encoded by the adenoviral entity on that molecule.

[0297] All publications and patent applications mentioned inthis specification are indicative of the level of skill of those skilled in the art to which this invention pertains. All publications and patent applications are herein incorporated by reference to the same extent as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference.

[0298] The invention now having been fully described, it will be apparent to one of ordinary skill in the art that many changes and modifications can be made thereto without departing from the spirit or scope of the appended claims. TABLE 1 Primers used for PCR amplification of DNA fragments used for generation of constructs described in this patent application. Ea-1 CGTGTAGTGTATTTATACCCG PCR amplification Ad5 nt.459 -> (SEQ. I.D. NO. 1) Ea-2 TCGTCACTGGGTGGAAAGCCA PCR amplification Ad5 nt.960 <- (SEQ. I.D. NO. 2) Ea-3 TACCCGCCGTCCTAAAATGGC nt.1284-1304 of Ad5 genome (SEQ. I.D. NO. 3) Ea-5 TGGACTTGAGCTGTAAACGC nt.1514-1533 of Ad5 genome (SEQ. I.D. NO. 4) Ep-2 GCCTCCATGGAGGTCAGATGT nt.1721-1702 of Ad5; introduction of Nco (SEQ. I.D. NO. 5) I site Eb-1 GCTTGAGCCCGAGACATGTC nt.3269-3289 of AdS genome (SEQ. I.D. NO. 6) Eb-2 CCCCTCGAGCTCAATCTGTATCTT nt.3508-3496 of Ad5 genome; (SEQ. I.D. NO. 7) introduction of Xho I site SV40-1 GGGGGATCCGAACTTGTTTATTGCAGC introduction BamH I site (nt.2182-2199 of (SEQ. I.D. NO. 8) pMLP.TK) adaption of recombinant adenoviruses SV40-2 GGGAGATCTAGACATGATAAGATAC introduction Bgl II site (nt.2312-2297 of (SEQ. I.D. NO. 9) pMLP.TK) Ad5-1 GGGAGATCTGTACTGAAATGTGTGGGC introduction Bgl II site (nt.2496-2514 of (SEQ. I.D. NO. 10) pMLP.TK) Ad5-2 GGAGGCTGCAGTCTCCAACGGCGT nt.2779-2756 of pMLP.TK (SEQ. I.D. NO. 11) ITR1 GGGGGATCCTCAAATCGTCACTTCCGT nt.35737-35757 of Ad5 (introduction of (SEQ. I.D. NO. 12) BamH I site ITR2 GGGGTCTAGACATCATCAATAATATAC nt.35935-35919 of Ad5 (introduction of (SEQ. I.D. NO. 13) XbaI site) PCR primer sets to be used to create the Sal I and Asp 718 sites juxtaposed to the ITR sequences. PCR/MLP1 GGCGAATTCGTCGACATCATCAATAATATACC (SEQ. I.D. NO. 14) (Ad5 nt.10-18) PCR/MLP2 GGCGAATTCGGTACCATCATCAATAATATACC (SEQ. I.D. NO. 15) (Ad5 nt.10-18) PCR/MLP3 CTGTGTACACCGGCGCA (SEQ. I.D. NO. 16) (Ad5 nt.200-184) Synthetic oligonucleotide pairs used to generate a synthetic hairpin, recreates an Asp718 site at one of the termini if inserted in Asp718 site: HP/asp1 5′-GTACACTGACCTAGTGCCGCCCGGGAAAGCCCGGGCGGCACTAGGTCAG (SEQ. I.D. NO. 17) HP/asp2 5′-GTACCTGACCTAGTGCCGCCCGGGCTTTGCCCGGGCGGCACTAGGTCAGT (SEQ. I.D. NO. 18) Synthetic oligonucleotide pair used to generate a synthetic hairpin, contains the ClaI recognition site to be used for hairpin formation HP/cla1 5′-GTACATTGACCTAGTGCCGCCCGGGCAAAGCCCGGGCGGCACTAGGTCAATCGAT (SEQ. I.D. NO. 19) HP/cla2 5′-GTACATCGATTGACCTAGTGCCGCCCGGGTTTGCCCGGGCGGCACTAGGTCAAT (SEQ. I.D. NO. 20)

[0299] TABLE II Production of recombinant adenoviral vector or different packages, cell lines. Yields × 10⁻⁸ pfu/T175 flask.⁰⁰⁰¹ Cell Passage Number IG.Ad.CMV.lacZ IG.Ad.CMV.TK IG.Ad.MLPI.TK d1313 Producer Mean 293 6.0 5.8 24  34 17.5 911 8 14 34 180 59.5 PER.C3 17 8 11 44  40 25.8 PER.C5 15 6 17 36 200 64.7 PER.C6 36 10 22 58 320 102

[0300] TABLE III Double insert viruses with different transgenes replacing the E1 and E3/gp19K regions express both transgenes in human A549 cells. luciferase % of cells with activity Virus⁰⁰⁰² Amount HSA expression (light units) IGAd/CMV-luc 5 × 10⁷ i.u. ND 25,726,074 2.5 × 10⁷ i.u. ND  7,996,542 IGAd/S1800-HSA 100 μl ccl 88% ND 50 μl ccl 82% ND IGAd/S1800-HSA.E3luc 1.2 × 10⁷ i.u. 97% 32,451,300 6 × 10⁷ i.u. 97% 24,716,586 1.2 × 10⁸ i.u. 100%  13,294,321

[0301]

1 47 1 23 DNA Artificial Sequence Description of Artificial Sequence oligo linker 1 aattgtctta attaaccgct taa 23 2 19 DNA Artificial Sequence misc_feature (1)..(19) /Note=“linker with Pac site” 2 aattgtctta attaaccgc 19 3 19 DNA Artificial Sequence misc_feature (1)..(19) /Note=“linker with Pac site” 3 aattgcggtt aattaagac 19 4 47 DNA Artificial Sequence primer_bind (1)..(47) /Note=“primer LTR-1” 4 ctgtacgtac cagtgcactg gcctaggcat ggaaaaatac ataactg 47 5 64 DNA Artificial Sequence primer_bind (1)..(64) /Note=“primer LTR-2” 5 gcggatcctt cgaaccatgg taagcttggt accgctagcg ttaaccgggc gactcagtca 60 atcg 64 6 28 DNA Artificial Sequence primer_bind (1)..(28) /Note=“primer HSA1” 6 gcgccaccat gggcagagcg atggtggc 28 7 50 DNA Artificial Sequence primer_bind (1)..(50) /Note=“primer HSA2” 7 gttagatcta agcttgtcga catcgatcta ctaacagtag agatgtagaa 50 8 21 DNA Artificial Sequence primer_bind (1)..(21) /Note=“primer1” 8 gggtattagg ccaaaggcgc a 21 9 33 DNA Artificial Sequence primer_bind (1)..(33) /Note=“primer 2” 9 gatcccatgg aagcttgggt ggcgacccca gcg 33 10 36 DNA Artificial Sequence primer_bind (1)..(36) /Note=“primer 3” 10 gatcccatgg ggatccttta ctaagttaca aagcta 36 11 19 DNA Artificial Sequence primer_bind (1)..(19) /Note=“primer 4” 11 gtcgctgtag ttggactgg 19 12 25 DNA Artificial Sequence primer_bind (1)..(25) /Note=“primer” 12 cgataagctt aattcctttg tgttt 25 13 33 DNA Artificial Sequence primer_bind (1)..(33) /Note=“primer” 13 cttaggtaac ccagtagatc cagaggagtt cat 33 14 63 DNA Artificial Sequence primer_bind (1)..(63) /Note=“primer polyL-ITR” 14 aactgcagat ctatcgatac tagtcaattg ctcgagtcta gactacgtca cccgccccgt 60 tcc 63 15 39 DNA Artificial Sequence primer_bind (1)..(39) /Note=“primer ITR-BSN” 15 cgggatccgt cgacgcggcc gcatcatcaa taatatacc 39 16 10 DNA Artificial Sequence misc_feature (1)..(10) /Note=“phosphorylated NSI linker” 16 cgatgcatcg 10 17 32 DNA Artificial Sequence primer_bind (1)..(32) /Note=“primer 1” 17 ggggtggcca gggtacctct aggcttttgc aa 32 18 29 DNA Artificial Sequence primer_bind (1)..(29) /Note=“primer 2” 18 ggggggatcc ataaacaagt tcagaatcc 29 19 21 DNA Artificial Sequence misc_feature (1)..(21) /Note=“linker” 19 agcttgaatt cccgggtacc t 21 20 21 DNA Artificial Sequence misc_feature (1)..(21) /Note=“linker” 20 ctagaggtac ccgggaattc a 21 21 39 DNA Artificial Sequence primer_bind (1)..(39) /Note=“primer ITR-EPH” 21 cggaattctt aattaagtta acatcatcaa taatatacc 39 22 66 DNA Artificial Sequence primer_bind (1)..(66) /Note=“primer ITR-pIX” 22 acggcgcgcc ttaagccacg cccacacatt tcagtacgta ctagtctacg tcacccgccc 60 cgttcc 66 23 26 DNA Artificial Sequence primer_bind (1)..(26) /Note=“primer Ad3′/Forw” 23 cggaattcat caggataggg cggtgg 26 24 44 DNA Artificial Sequence primer_bind (1)..(44) /Note=“primer Ad3′/Rev” 24 cgggatccta tcgatattta aatgttttag ggcggagtaa cttg 44 25 37 DNA Artificial Sequence primer_bind (1)..(37) /Note=“primer PA-pIX1” 25 taagccacta gtacgtactg aaatgtgtgg gcgtggc 37 26 43 DNA Artificial Sequence primer_bind (1)..(43) /Note=“primer PA-pIX2” 26 ttaagccacg cccacacatt tcagtacgta ctagtggctt aat 43 27 21 DNA Artificial Sequence primer_bind (1)..(21) /Note=“primer Ea-1” 27 cgtgtagtgt atttataccc g 21 28 21 DNA Artificial Sequence primer_bind (1)..(21) /Note=“primer Ea-2” 28 tcgtcactgg gtggaaagcc a 21 29 21 DNA Artificial Sequence primer_bind (1)..(21) /Note=“primer Ea-3” 29 tacccgccgt cctaaaatgg c 21 30 20 DNA Artificial Sequence primer_bind (1)..(20) /Note=“primer Ea-5” 30 tggacttgag ctgtaaacgc 20 31 21 DNA Artificial Sequence primer_bind (1)..(21) /Note=“primer Ep-2” 31 gcctccatgg aggtcagatg t 21 32 20 DNA Artificial Sequence primer_bind (1)..(20) /Note=“primer Eb-1” 32 gcttgagccc gagacatgtc 20 33 24 DNA Artificial Sequence primer_bind (1)..(24) /Note=“primer Eb-2” 33 cccctcgagc tcaatctgta tctt 24 34 27 DNA Artificial Sequence primer_bind (1)..(27) /Note=“primer SV40-1” 34 gggggatccg aacttgttta ttgcagc 27 35 25 DNA Artificial Sequence primer_bind (1)..(25) /Note=“primer SV40-2” 35 gggagatcta gacatgataa gatac 25 36 27 DNA Artificial Sequence primer_bind (1)..(27) /Note=“primer Ad5-1” 36 gggagatctg tactgaaatg tgtgggc 27 37 24 DNA Artificial Sequence primer_bind (1)..(24) /Note=“primer Ad5-2” 37 ggaggctgca gtctccaacg gcgt 24 38 27 DNA Artificial Sequence primer_bind (1)..(27) /Note=“primer ITR1” 38 gggggatcct caaatcgtca cttccgt 27 39 27 DNA Artificial Sequence primer_bind (1)..(27) /Note=“primer ITR2” 39 ggggtctaga catcatcaat aatatac 27 40 32 DNA Artificial Sequence primer_bind (1)..(32) /Note=“primer PCR/MLP1” 40 ggcgaattcg tcgacatcat caataatata cc 32 41 32 DNA Artificial Sequence primer_bind (1)..(32) /Note=“primer PCR/MLP2” 41 ggcgaattcg gtaccatcat caataatata cc 32 42 17 DNA Artificial Sequence primer_bind (1)..(17) /Note=“primer PCR/MLP3” 42 ctgtgtacac cggcgca 17 43 49 DNA Artificial Sequence primer_bind (1)..(49) /Note=“primer HP/asp1” 43 gtacactgac ctagtgccgc ccgggaaagc ccgggcggca ctaggtcag 49 44 50 DNA Artificial Sequence primer_bind (1)..(50) /Note=“primer HP/asp2” 44 gtacctgacc tagtgccgcc cgggctttgc ccgggcggca ctaggtcagt 50 45 55 DNA Artificial Sequence primer_bind (1)..(55) /Note=“primer HP/cla1” 45 gtacattgac ctagtgccgc ccgggcaaag cccgggcggc actaggtcaa tcgat 55 46 54 DNA Artificial Sequence primer_bind (1)..(54) /Note=“primer HP/cla2” 46 gtacatcgat tgacctagtg ccgcccgggt ttgcccgggc ggcactaggt caat 54 47 45 DNA Artificial Sequence misc_feature (1)..(45) /Note=“hac, haw Potential hairpin that can be formed after digestion with restriction endonuclease Asp7I8 in both the correct and in the reverse orientation, respectively” 47 gtacactgac ctagtgccgc ccgggcaaag cccgggcggc actag 45 

What is claimed is:
 1. A recombinant nucleic acid comprising an adenoviral clone, said adenoviral clone selected from the group consisting of pBr/Ad.Bam-rITR (ECACC P97082122), pBr/Ad.Sal-rITR (ECACC P97082119), pBr/Ad.Cla-Bam (ECACC P97082117), uBr/Ad.Afl II-Bam (ECACC P97082114), pBr/Ad.Bam-rITRpac#2 (ECACC P97082120), pBr/Ad.Bam-rITR#8 (ECACC P97082121), pWE/Ad.AflII-rITR (ECACC P97082116), pBr/Ad.1ITR-Sal(9.4) (ECACC P97082115), and pBr/Ad. 1ITR-Sal(16.7)(ECACC P97082118).
 2. The recombinant nucleic acid of claim 1, further comprising a deletion in an E3 region.
 3. The recombinant nucleic acid of claim 2, wherein said deletion in the E3 region is replaced with a transgene.
 4. The recombinant nucleic acid of claim 1, further comprising a deletion in a gp19K region.
 5. The recombinant nucleic acid of claim 4, wherein the deletion in the gp19K region is replaced with a transgene.
 6. A recombinant nucleic acid comprising an adapter plasmid, said adapter plasmid selected from the group consisting of pWE/Ad.AflIII-EcoRI, pAd5/CLIP, pAd5/L420-HSA, pMV/L420-H, pMV/CMV-LacZ, and pWE/Ad-H.
 7. The recombinant nucleic acid of claim 6, wher ein said adapter plasmid is pAd5/CLIP or pAd5/L420-HSA, and said recombinant nucleic acid comprises adenovirus derived nucleotides 1-454; and adenovirus nucleotides 3511-6095.
 8. A recombinant nucleic acid comprising a recombinant adenovirus, wherein said recombinant adenovirus is pBS.Eco-Eco/ad5ΔHIIIΔgp19KΔXbaI.
 9. The recombinant nucleic acid of claim 8, wherein said nucleic acid further comprises a transgene.
 10. The recombinant nucleic acid of claim 9, wherein said transgene is operatively linked to an E3 promoter.
 11. The recombinant nucleic acid of claim 9, wherein said transgene is selected from the group consisting of a suicide gene, a cytokine gene, and a reporter gene.
 12. The recombinant nucleic acid of claim 11, wherein said transgene is a suicide gene comprising an HSV-TK gene.
 13. The recombinant nucleic acid of claim 11, wherein said transgene comprises a coding sequence selected from the group consisting of hIL-1α, rat IL-3, and human IL-3.
 14. The recombinant nucleic acid of claim 11, wherein said transgene comprises a coding sequence from a luciferase gene or a hacZ gene.
 15. The recombinant nucleic acid of claim 14, further comprising a heterologous nucleotide sequence.
 16. The recombinant nucleic acid of claim 9, wherein said transgene comprises a coding sequence from a human ceNOS gene.
 17. A recombinant nucleic acid comprising: an adenoviral nucleotide sequence, said adenoviral nucleotide sequence comprising a functional encapsulating signal and two functional Inverted Terminal Repeats or functional fragments or derivatives thereof acting as Inverted Terminal Repeats, said recombinant nucleic acid lacking functional adenoviral genes and having no overlapping sequences allowing for homologous recombination leading to replication competent virus in a cell into which said recombinant nucleic acid is transferred.
 18. A recombinant nucleic acid for incorporation into a cell comprising: an adenoviral nucleotide sequence comprising sufficient adenoviral sequences necessary for replication and capsid gene expression, but lacking at least the E1 region and an adenoviral encapsulating signal, wherein said adenoviral nucleotide sequence further lacks overlapping sequences allowing for homologous recombination leading to replication competent virus in the cell into which said recombinant nucleic acid is to be transferred.
 19. The recombinant nucleic acid of claim 18, further comprising a deletion in an E3 region of said recombinant nucleic acid.
 20. The recombinant nucleic acid of claim 18, further comprising a deletion in a gp19K region of said recombinant nucleic acid
 21. A recombinant nucleic acid comprising a construct comprising pWE/Ad.Δ5′.
 22. The recombinant nucleic acid of claim 21, further comprising a deletion in an E3 region of said recombinant nucleic acid.
 23. The recombinant nucleic acid of claim 21, further comprising a deletion in a gp19K region of said recombinant nucleic acid.
 24. A recombinant nucleic acid comprising: an adenoviral nucleotide sequence comprising sufficient adenovirus sequences needed for replication and capsid gene expression and a complementary sequence to an upstream part of said recombinant nucleic acid, wherein said complementary sequence can base-pair with said upstream part so that it functions as a start-site for a nucleic acid polymerase, said adenoviral nucleotide sequence having a deletion of one Inverted Terminal Repeat, the E1 region, and the adenoviral encapsulating signal, wherein said recombinant nucleic acid has no overlapping sequences allowing for homologous recombination leading to replication competent virus in a cell into which said recombinant nucleic acid is transferred.
 25. The recombinant nucleic acid according to claim 25, wherein said recombinant nucleic acid comprises a construct pWE/AAV.Δ5′.
 26. A recombinant nucleic acid comprising: an adenoviral nucleotide sequence comprising a sequence for adenovirus-independent replication and sufficient adenoviral sequences necessary for replication; wherein said adenoviral nucleotide sequence has a deletion of at least the E1 region and encapsulating signal of adenovirus, said recombinant nucleic acid comprising no overlapping sequences which allow for homologous recombination leading to replication competent virus in a cell into which said recombinant nucleic acid is transferred.
 27. The recombinant nucleic acid of claim 26, wherein said adenoviral nucleotide sequence further has a deletion of at least one of the Inverted Terminal Repeats of said adenovirus.
 28. The recombinant nucleic acid of claim 26, wherein said adenoviral nucleotide sequence for adenovirus-independent replication comprises an SV40 origin of replication.
 29. An adapter plasmid comprising: an adenoviral nucleotide sequence comprising, in operable configuration, at least one functional Inverted Terminal Repeat and one functional encapsulating signal, said adapter plasmid having no sequences which allow for homologous recombination leading to replication competent virus in a cell into which said adapter plasmid is transferred.
 30. The adapter plasmid of claim 29, wherein said adapter plasmid has no E1 region sequences.
 31. The adapter plasmid of claim 29, further comprising a multiple cloning site.
 32. The adapter plasmid of claim 31, further comprising a nucleic acid inserted into said multiple cloning site.
 33. A method of generating a recombinant adenovirus having an E1 deletion and a gp19K deletion, said method comprising: growing a cell comprising adenovirus complementing sequences transfected with: i) an adapter plasmid comprising a first nucleotide sequence based on or derived from an adenovirus, wherein said nucleotide sequence comprises in operable configuration one functional Inverted Terminal Repeat, one functional encapsulating signal and adenoviral sequences which allow for homologous recombination leading to the generation of a replication-defective, recombinant adenovirus genome in a cell into which said adapter plasmid is transferred and having no E1 region sequences, and ii) a recombinant nucleic acid comprising at least one second nucleotide sequence based on or derived from an adenovirus, wherein said at least one second nucleotide sequence comprises one Inverted Terminal Repeat and sufficient adenovirus sequences for replication and a partial overlap with said adapter plasmid, wherein said at least one second nucleotide sequence comprises a deletion of at least the E1 region, encapsulating signal and gp19K sequences; wherein said complementing sequences, said first nucleotide sequence and said at least one second nucleotide sequence have no overlapping sequences which allow for homologous recombination leading to replication competent virus, under conditions wherein recombinant adenovirus having an E1 deletion and a gp19K deletion is generated.
 34. The method according to claim 33, wherein said adapter plasmid further comprises a first heterologous nucleotide sequence inserted into said E1 region deletion and said recombinant nucleic acid further comprises a second heterologous nucleotide sequence inserted into said gp19K region.
 35. The method according to claim 33, wherein said at least one second nucleotide sequence comprises a first and second molecule wherein said first molecule has said partial overlap with said adapter plasmid at the 3′ end, and said second molecule comprises said Inverted Terminal Repeat and region including deletion of said gp19K sequences.
 36. A method of generating a recombinant adenovirus, said method comprising: growing a cell comprising adenovirus complementing sequences transfected with: i) a first recombinant nucleic acid comprising a first nucleotide sequence based on or derived from an adenovirus, wherein said first nucleotide sequence comprises a functional encapsulating signal and two functional Inverted Terminal Repeats or functional fragments or derivatives thereof, and wherein said first recombinant nucleic acid has no functional adenoviral genes and ii) a second recombinant nucleic acid comprising a second nucleotide sequence based on or derived from an adenovirus, wherein said second nucleotide sequence comprises sufficient adenovirus sequences for replication, wherein said second nucleotide sequence comprises a deletion of at least the E1 region and encapsulating signal of said adenovirus; wherein, said complementing sequences, said first nucleotide sequence and said second nucleotide sequence have no overlapping sequences which allow for homologous recombination leading to replication competent virus, under conditions wherein recombinant adenovirus is generated.
 37. A method of generating a recombinant adenovirus, said method comprising: growing a cell comprising adenovirus complementing sequences transfected with: i) a first recombinant nucleic acid comprising a first nucleotide sequence based on or derived from an adenovirus, wherein said first nucleotide sequence comprises a functional encapsulating signal and two functional Inverted Terminal Repeats or functional fragments or derivatives thereof, and wherein said first recombinant nucleic acid has no functional adenoviral genes and ii) a second recombinant nucleic acid comprising a second nucleotide sequence based on or derived from an adenovirus, wherein said second nucleotide sequence comprises a sequence for adenovirus-independent replication, and sufficient adenoviral sequences necessary for replication, wherein said second nucleotide sequence comprises at least a deletion of the E1 region and encapsulating signal of said adenovirus; wherein, said complementing sequences, said first nucleotide sequence and said second nucleotide sequence have no overlapping sequences which allow for homologous recombination leading to replication competent virus, under conditions wherein recombinant adenovirus is generated.
 38. The method according to claim 37, wherein said cell comprises at least one nucleic acid molecule and wherein said cell expresses SV40 Large T antigen proteins or functional fragments of.SV40 Large T antigen proteins having the activity thereof.
 39. The method according to claim 38, wherein said second recombinant nucleic acid molecule is replicated.
 40. A replication defective adenovirus comprising: an adenoviral genome comprising at least a functional encapsulating signal and two functional Inverted Terminal Repeats or fragments or derivatives thereof having the activity of a functional Inverted Terminal Repeat, and lacking functional adenoviral genes and having no overlapping sequences allowing for homologous recombination leading to replication competent virus in a cell into which said replication defective adenovirus is transferred.
 41. The replication defective adenovirus of claim 40, wherein the adenoviral genome further comprises at least one expression cassette.
 42. The replication defective adenovirus of claim 41, wherein said at least one expression cassette comprises a gene functionally linked to transcription regulatory sequences.
 43. The replication defective adenovirus of claim 40, further comprising one or more non-adenoviral nucleic acid sequences.
 44. The replication defective adenovirus of claim 43, wherein said one or more non-adenoviral nucleic acid sequences are inserted into the E1 region or into the E3 region gp19K gene.
 45. A cell comprising: a replication defective adenovirus comprising an adenoviral genome of a replication defective adenovirus, said adenoviral genome having at least a functional encapsulating signal and two functional Inverted Terminal Repeats or fragments or derivatives thereof having the activity of a functional Inverted Terminal Repeat, said adenoviral genome lacking functional adenoviral genes and having no overlapping sequences allowing for homologous recombination leading to replication competent virus in said cell into which said replication defective adenovirus has been transferred.
 46. The cell of claim 45, wherein said cell is a mammalian cell.
 47. A method of generating recombinant adenovirus incapable ofreplicating, said method comprising: growing a cell comprising adenovirus complementing sequences and i) a first recombinant nucleic acid comprising a first nucleotide sequence based on or derived from an adenovirus, wherein said first nucleotide sequence comprises a functional encapsulating signal and two functional Inverted Terminal Repeats or functional fragments or derivatives thereof, and wherein said first recombinant nucleic acid has no functional adenoviral genes and, ii) a second recombinant nucleic acid comprising a second nucleotide sequence based on or derived from an adenovirus, wherein said nucleotide sequence comprises at least all adenovirus sequences or functional fragments or derivatives thereof necessary for replication and capsid gene expression, and a complementary sequence to an upstream part of the same strand of said nucleic acid, wherein said complementary sequence can base-pair with said upstream part so that it functions as a start-site for a nucleic acid polymerase, wherein said second nucleotide sequence comprises a deletion of one Inverted Terminal Repeat, the E1 region and the encapsulating signal of said adenovirus; wherein, said complementing sequences, said first nucleotide sequence and said second nucleotide sequence have no overlapping sequences which allow for homologous recombination leading to replication competent virus, under conditions wherein recombinant adenovirus is generated.
 48. A cell comprising the recombinant nucleic acid of claim
 1. 49. A method of replacing a defective gene in a genome of a host cell, said method comprising: growing said host cell with a recombinant nucleic acid molecule derived from a replication defective adenovirus comprising a functional version or part of said defective gene under conditions wherein at least one allele of said defective gene in said host cell genome is replaced.
 50. The method according to claim 49, wherein said replication defective adenovirus expresses no adenoviral genes.
 51. The method according to claim 49, wherein said defective gene is a defective tumor suppressor gene.
 52. An isolated cell comprising: an adenoviral genome of a replication defective adenovirus, said adenoviral genome having at least a functional encapsulating signal and two functional Inverted Terminal Repeats or fragments or derivatives thereof having the activity of a functional Inverted Terminal Repeat, said adenoviral genome lacking functional adenoviral genes and having no overlapping sequences allowing for homologous recombination leading to replication competent virus in said isolated cell.
 53. The isolated cell of claim 52, wherein said cell is a human cell.
 54. A replication-defective adenovirus comprising an adenoviral genome, wherein said adenoviral genome has a first deletion in an adenoviral E1 region and a second deletion in an adenoviral gp19K region.
 55. The replication-defective adenovirus of claim 54, wherein the adenoviral genome further comprises a transgene.
 56. The replication-defective adenovirus of claim 55, wherein transcription of said transgene is directed by an E3 promoter.
 57. A cell comprising the recombinant nucleic acid of claim
 17. 58. A cell comprising the recombinant nucleic acid of claim
 24. 59. A cell comprising the recombinant nucleic acid of claim
 26. 